Asymmetric auxiliary group

ABSTRACT

To provide a chiral reagent or a salt thereof. The chiral reagent has following chemical formula (I). In the formula (I), G 1  and G 2  are independently a hydrogen atom, a nitro group (—NO 2 ), a halogen atom, a cyano group (—CN), a group of formula (II) or (III), or both G 1  and G 2  taken together to form a group of formula (IV).

FIELD OF THE INVENTION

The present invention is directed to a chiral reagent that is used tosynthesize stereocontrolled phosphorus atom-modified oligonucleotidederivatives.

BACKGROUND OF THE INVENTION

JP 2005-89441 A discloses a method for producing a derivative ofnucleotides called an oxazaphospholidine method. However, the isolateyield of the monomers is low and the method requires special cappingagents that are not commercially available. Further obtained monomersare chemically unstable. Furthermore, the isolate yields ofoligonucleotide derivatives are not high. It is thought that the lowyield of oligonucleotide derivatives is caused by the degradationreactions under the de-protection steps.

WO2010/064146 pamphlet discloses a method for producing a derivative ofnucleotides. The method disclosed therein requires special cappingagents that are not commercially available. Furthermore, the isolateyields of oligonucleotide derivatives are not high. The low yield isthought to be caused by the degradation reactions under thede-protection steps. This tendency becomes strongly apparent when thelength of oligonucleotide derivatives becomes long.

WO2012/039448 pamphlet discloses Asymmetric auxiliary group which isused to produce stereocontrolled phosphorus atom-modifiedoligonucleotide derivatives.

CITATION LIST Patent Literature

[Patent Literature 1] JP 2005-89441 A

[Patent Literature 2] WO2010/064146 A

[Patent Literature 3] WO2012/039448 A

SUMMARY OF THE INVENTION

The first Aspect of the Invention relates to a chiral reagent or a saltthereof. The chiral reagent has following chemical formula (I).

In the formula (I), G¹ and G² are independently a hydrogen atom, a nitrogroup (—NO₂), a halogen atom, a cyano group (—CN), a group of formula(II), (III) or (V), or both G¹ and G² taken together to form a group offormula (IV).

In the formula (II), G²¹ to G²³ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group or C₁₋₃ alkyl group.

In the formula (III), G³¹ to G³³ are independently C₁₋₄ alkyl group,C₆₋₁₄ aryl group C₁₋₄ alkoxy group, C₇₋₁₄ aralkyl group, C₁₋₄ alkylC₆₋₁₄ aryl group, C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkylgroup.

In the formula (IV), G⁴¹ to G⁴⁶ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group or C₁₋₃ alkyl group.

In the formula (V), G⁵¹ to G⁵³ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group, C₁₋₃ alkyl group or C₁₋₃alkyloxy group.

G³ and G⁴ are independently a hydrogen atom, C₁₋₃ alkyl group, C₆₋₁₄aryl group, or both G³ and G⁴ taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms, together withthe NH moiety in formula (I).

A preferred embodiment is that the chiral reagent has following chemicalformula (I′).

In the formula (I′), G¹ and G² are same as above. Namely, G¹ and G² areindependently a hydrogen atom, a nitro group, a halogen atom, a cyanogroup, a group of formula (II) or (III), or both G¹ and G² takentogether to form a group of formula (IV).

A preferred embodiment is that the chiral reagent has chemical formula(I′) and each of G¹ and G² is a group of formula (II), wherein G²¹ toG²³ are independently a hydrogen atom, a nitro group, a halogen atom, acyano group or C₁₋₃ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and each of G¹ and G² is a group of formula (II) and each of G²¹ toG²³ is a hydrogen atom

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (II), and G²¹to G²³ are independently a hydrogen atom, a nitro group, a halogen atom,a cyano group or C₁₋₃ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (II), each ofG²¹ and G²² is a hydrogen atom and G²³ is a nitro group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom and G² is a group of formula (III), andG³¹ to G³³ are independently C₁₋₄ alkyl group, C₆₋₁₄ aryl group, C₇₋₁₄aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group, C₁₄ alkoxy C₆₋₁₄ aryl group,or C₆₋₁₄ aryl C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom and G² is a group of formula (III), andG³¹ to G³³ are independently C 14 alkyl group, C₆ aryl group, C₇₋₁₀aralkyl group, C₁₋₄ alkyl C₆ aryl group, C₁₋₄ alkoxy C₆ aryl group, orC₆ aryl C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (III), and G³¹to G³³ are independently C₁₋₄ alkyl group or C₆ aryl group. Examples ofC₁₋₄ alkyl group are methyl group, ethyl group, n-propyl group,iso-propyl group, n-buthyl group and tert-buthyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (III), and G³¹to G³³ are independently C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (III), and G³¹and G³³ are C₆ aryl group and G³² is C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ and G² taken together to form a group of formula (IV), andG⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, a halogenatom, a cyano group or C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ and G² taken together to form a group of formula (IV),wherein each of G⁴¹ to G⁴⁶ is a hydrogen atom.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom and G² is a group of formula (V). Furthereach of G⁵¹ to G⁵³ is independently a hydrogen atom, a nitro group, amethyl group, or a methoxy group. More preferred embodiment is that G¹is a hydrogen atom and G² is a group of formula (V), wherein each of G″and G⁵³ is a hydrogen atom and G⁵³ is a 4-methyl group.

A preferred embodiment is that the chiral reagent is selected from oneof III-a, III-b,

V-a, VII-a, VII-b, IX-a, IX-b, XI-a, XIII-a and XIII-b:(S)-2-(Methyldiphenylsilyl)-1-((S)-pyrrolidin-2-yl)ethanol (III-a)

-   (R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol (III-b)-   (S)-2-(Trimethylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (V-a)-   (R)-2,2-Diphenyl-1-((S)-pyrrolidin-2-yl)ethanol (VII-a)-   (S)-2,2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (VII-b)-   (R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (IX-a)-   (S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (IX-b)-   (R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (XI-a)-   (S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (XIII-a)-   (R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (XIII-b)

The second aspect of the invention relates to a nucleoside3′-phosphoramidite derivative which is represented by formula (Va) or(Vb).

In the formula (Va) and (Vb), G¹ to G⁴ are same as above, G⁵ is aprotective group of the hydroxyl group, and Bs is a group selected fromthe groups represented by following formula (VI) to (XI) or derivativesthereof.

Examples of Bs are an adenine, a thymine, a cytosine, a guanine, anuracil, a 5-methylcytosine or derivative thereof.

R² is hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen, alkyl, alkenyl,alkynyl, alkyl-Y¹-, alkenyl-Y¹-, alkynyl-Y¹-, aryl-Y¹-, heteroaryl-Y¹-,—OR^(b), or —SR^(b), wherein R^(b) is a blocking moiety.

Y¹ is O, NR^(d), S, or Se.

R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,substituted silyl, carbamate, —P(O)(Re)₂, or —HP(O)(R^(e)).

R^(e) is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or acation which is Na⁺, Li⁺ or K⁺.

Y² is O, NR^(d), or S.

R³ is a group represented by —CH₂—, —(CH₂)₂—, —CH₂NH—, or —CH₂N(CH₃)—.

Examples of G⁵ are trityl, 4-monomethoxytrityl, 4,4′-dimethoxytrityl,4,4′,4″-trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX).

A preferred embodiment of the second aspect is that the nucleoside3′-phosphoramidite derivative is represented by formula (Va′) or (Vb′).

In the formula (Va′) and (Vb′), G¹, G², G⁵, Bs, R², and R³ are same asabove.

The third aspect of the invention relates to a method for synthesis of astereocontrolled phosphorus atom-modified oligonucleotide derivative.

First step is a step of reacting a molecule comprising an achiralH-phosphonate moiety, the first activating reagent and a chiral reagentor a salt thereof to form a monomer. The chiral reagent has chemicalformula (I) or (I′) and the monomer may be represented by formula (Va),(Vb), (Va′), or (Vb′). The monomer reacts with the second activatingreagent and a nucleoside to form a condensed intermediate. Next step isa step of converting the condensed intermediate to the nucleic acidcomprising a chiral X-phosphonate moiety.

Based on the present method, it is possible to use stable andcommercially available materials as starting materials. It is possibleto produce stereocontrolled phosphorus atom-modified oligonucleotidederivatives using an achiral starting material.

As shown in a working example, the method of the present invention doesnot cause degradations under de-protection steps. Further the methoddoes not require special capping agents to produce phosphorusatom-modified oligonucleotide derivatives.

The fourth aspect of the invention relates to a method for synthesis ofstereocontrolled phosphorus atom-modified oligonucleotide derivativesusing a chiral monomer.

The first step is reacting a nucleoside 3′-phosphoramidite derivativewhich is represented by formula (Va), (Vb), (Va′), or (Vb′) with thesecond activating reagent and a nucleoside to form a condensedintermediate. The second step is converting the condensed intermediateto the nucleic acid comprising a chiral X-phosphonate moiety.

INCORPORATION BY REFERENCE

All publications and patent applications disclosed herein in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is UPLC profile in producing oligonucleotide derivative using themonomer of 4b.

FIG. 2 is UPLC profile in producing oligonucleotide derivative using themonomer of 25.

BEST MODE FOR CARRYING OUT THE INVENTION

The term “nucleic acid” encompasses poly- or oligo-ribonucleotides (RNA)and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived fromN-glycosides or C-glycosides of nucleobases and/or modified nucleobases;nucleic acids derived from sugars and/or modified sugars; and nucleicacids derived from phosphate bridges and/or modified phosphorus-atombridges. The term encompasses nucleic acids containing any combinationsof nucleobases, modified nucleobases, sugars, modified sugars, phosphatebridges or modified phosphorus atom bridges. Examples include, and arenot limited to, nucleic acids containing ribose moieties, the nucleicacids containing deoxyribose moieties, nucleic acids containing bothribose and deoxyribose moieties, nucleic acids containing ribose andmodified ribose moieties. The prefix poly- refers to a nucleic acidcontaining about 1 to about 10,000 nucleotide monomer units and whereinthe prefix oligo- refers to a nucleic acid containing about 1 to about200 nucleotide monomer units.

The term “nucleobase” refers to the parts of nucleic acids that areinvolved in the hydrogen-bonding that binds one nucleic acid strand toanother complementary strand in a sequence specific manner. The mostcommon naturally-occurring nucleobases are adenine (A), guanine (G),uracil (U), cytosine (C), 5-methylcytosine, and thymine (T).

The term “modified nucleobase” refers to a moiety that can replace anucleobase. The modified nucleobase mimics the spatial arrangement,electronic properties, or some other physicochemical property of thenucleobase and retains the property of hydrogen-bonding that binds onenucleic acid strand to another in a sequence specific manner. A modifiednucleobase can pair with all of the five naturally occurring bases(uracil, thymine, adenine, cytosine, or guanine) without substantiallyaffecting the melting behaviour, recognition by intracellular enzymes oractivity of the oligonucleotide duplex.

The term “nucleoside” refers to a moiety wherein a nucleobase or amodified nucleobase is covalently bound to a sugar or modified sugar.

The term “sugar” refers to a monosaccharide in closed and/or open form.Sugars include, but are not limited to, ribose, deoxyribose,pentofuranose, pentopyranose, and hexopyranose moieties.

The term “modified sugar” refers to a moiety that can replace a sugar.The modified sugar mimics the spatial arrangement, electronicproperties, or some other physicochemical property of a sugar.

The term “nucleotide” refers to a moiety wherein a nucleobase or amodified nucleobase is covalently linked to a sugar or modified sugar,and the sugar or modified sugar is covalently linked to a phosphategroup or a modified phosphorus-atom moiety.

The term “chiral reagent” refers to a compound that is chiral orenantiopure and can be used for asymmetric induction in nucleic acidsynthesis.

The term “chiral ligand” or “chiral auxiliary” refers to a moiety thatis chiral or enantiopure and controls the stereochemical outcome of areaction.

In a condensation reaction, the term “activating reagent” refers to areagent that activates a less reactive site and renders it moresusceptible to attack by a nucleophile.

The term “blocking moiety” refers to a group that transiently masks thereactivity of a functional group. The functional group can besubsequently unmasked by removal of the blocking moiety.

The terms “boronating agents”, “sulfur electrophiles”, “seleniumelectrophiles” refer to compounds that are useful in the modifying stepused to introduce BH₃, S, and Se groups, respectively, for modificationat the phosphorus atom.

The term “moiety” refers to a specific segment or functional group of amolecule.

Chemical moieties are often recognized chemical entities embedded in orappended to a molecule.

The term “solid support” refers to any support which enables syntheticmass production of nucleic acids and can be reutilized at need. As usedherein, the term refers to a polymer that is insoluble in the mediaemployed in the reaction steps performed to synthesize nucleic acids,and is derivatized to comprise reactive groups.

The term “linking moiety” refers to any moiety optionally positionedbetween the terminal nucleoside and the solid support or between theterminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refers to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect,” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit as described above. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylmoiety may be a saturated alkyl group (which means that it does notcontain any units of unsaturation, e.g. carbon-carbon double bonds orcarbon-carbon triple bonds) or the alkyl moiety may be an unsaturatedalkyl group (which means that it contains at least one unit ofunsaturation). The alkyl moiety, whether saturated or unsaturated, maybe branched, straight chain, or include a cyclic portion. The point ofattachment of an alkyl is at a carbon atom that is not part of a ring.

The “alkyl” moiety may have 1 to 10 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 10” refers to each integer inthe given range; e.g., “1 to 10 carbon atoms” means that the alkyl groupmay consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., upto and including 10 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). Alkyl includes both branched and straight chain alkylgroups. The alkyl group of the compounds described herein may bedesignated as “C₁-C₆ alkyl” or similar designations. By way of exampleonly, “C₁-C₆ alkyl” indicates that there are one, two, three, four,five, or six carbon atoms in the alkyl chain, i.e., the alkyl chain isselected from the group consisting of methyl, ethyl, propyl, iso-propyl,n-butyl, iso-butyl, sec-butyl, and tert-butyl. Typical alkyl groupsinclude, but are in no way limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tertiary butyl, pentyl, hexyl, allyl,cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, and the like. In one aspect, an alkyl is a C₁-C₆alkyl.

C₁₋₃ alkyl group means straight or branched alkyl group that has 1 to 3carbon atoms. Examples of C₁₋₃ alkyl group are methyl, ethyl, propyl andisopropyl. C₁₋₄ alkyl group means straight or branched alkyl group thathas 1 to 4 carbon atoms. Examples of C₁₋₄ alkyl group are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, and tert-butyl.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings are formed byfive, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups are a substituted or unsubstituted. In one aspect, an aryl is aphenyl or a naphthalenyl. Depending on the structure, an aryl group canbe a monoradical or a diradical (i.e., an arylene group). In one aspect,an aryl is a C₆-C₁₀ aryl.

C₆₋₁₄ aryl group means aryl group that has 6 to 14 carbon atoms. Theexamples of C₆₋₁₄ aryl group are phenyl, biphenyl, naphthyl, anthracyl,indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, andtetrahydronaphthyl.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

An “acyl moiety” refers to an alkyl(C═O), aryl(C═O), or aralkyl(C═O)group. An acyl moiety can have an intervening moiety (Y) that is oxy,amino, thio, or seleno between the carbonyl and the hydrocarbon group.For example, an acyl group can be alkyl-Y-(C═O), aryl-Y-(C═O) oraralkyl-Y-(C═O).

“Alkenyl” groups are straight chain, branch chain, and cyclichydrocarbon groups containing at least one carbon-carbon double bond.Alkenyl groups can be substituted.

“Alkynyl” groups are straight chain, branch chain, and cyclichydrocarbon groups containing at least one carbon-carbon triple bond.Alkynyl groups can be substituted.

An “alkoxy” group refers to an alklyl group linked to oxygen i.e.(alkyl)-O— group, where alkyl is as defined herein. Examples includemethoxy (—OCH₃) or ethoxy (—OCH₂CH₃) groups.

An “alkenyloxy” group refers to an alkenyl group linked to oxygen i.e.(alkenyl)-O— group, where alkenyl is as defined herein.

An “alkynyloxy” group refers to an alkynyl group linked to oxygen i.e.(alkynyl)-O— group, where alkynyl is as defined herein.

An “aryloxy” group refers to an aryl group linked to oxygen i.e.(aryl)-O— group, where the aryl is as defined herein. An exampleincludes phenoxy (—OC₆H₅) group.

The term “alkylseleno” refers to an alkyl group having a substitutedseleno group attached thereto i.e. (alkyl)-Se— group, wherein alkyl isdefined herein.

The term “alkenylseleno” refers to an alkenyl group having a substitutedseleno group attached thereto i.e. (alkenyl)-Se— group, wherein alkenylis defined herein.

The term “alkynylseleno” refers to an alkynyl group having a substitutedseleno group attached thereto i.e. (alkynyl)-Se— group, wherein alkenylis defined herein.

The term “alkylthio” refers to an alkyl group attached to a bridgingsulfur atom i.e. (alkyl)-S— group, wherein alkyl is defined herein. Forexample, an alkylthio is a methylthio and the like.

The term “alkenylthio” refers to an alkenyl group attached to a bridgingsulfur atom i.e. (alkenyl)-S— group, wherein alkenyl is defined herein.

The term “alkynylthio” refers to an alkynyl group attached to a bridgingsulfur atom i.e. (alkynyl)-S— group, wherein alkenyl is defined herein.

The term “alkylamino” refers to an amino group substituted with at leastone alkyl group i.e. —NH(alkyl) or —N(alkyl)₂, wherein alkyl is definedherein.

The term “alkenylamino” refers to an amino group substituted with atleast one alkenyl group i.e. —NH(alkenyl) or —N(alkenyl)₂, whereinalkenyl is defined herein.

The term “alkynylamino” refers to an amino group substituted with atleast one alkynyl group i.e. —NH(alkynyl) or —N(alkynyl)₂, whereinalkynyl is defined herein.

The term “halogen” is intended to include fluorine, chlorine, bromineand iodine.

A “fluorescent group” refers to a molecule that, when excited with lighthaving a selected wavelength, emits light of a different wavelength.Fluorescent groups include, but are not limited to, indole groups,fluorescein, tetramethylrhodamine, Texas Red, BODIPY,5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), coumarin andLucifer yellow.

An “ammonium ion” is a positively charged polyatomic cation of thechemical formula NH₄+.

An “alkylammonium ion” is an ammonium ion that has at least one of itshydrogen atoms replaced by an alkyl group, wherein alkyl is definedherein. Examples include triethylammonium ion,N,N-diisopropylethylammonium ion.

An “iminium ion” has the general structure R₂C=NR₂+. The R groups referto alkyl, alkenyl, alkynyl, aryl groups as defined herein. A“heteroaromatic iminium ion” refers to an imminium ion where thenitrogen and its attached R groups form a heteroaromatic ring. A“heterocyclic iminium ion” refers to an imminium ion where the nitrogenand its attached R groups form a heterocyclic ring.

The terms “amino” or “amine” refers to a —N(Rh)₂ radical group, whereeach Rh is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl, unless stated otherwise specifically inthe specification. When a —N(Rh)₂ group has two Rh other than hydrogenthey can be combined with the nitrogen atom to form a 4-, 5-, 6-, or7-membered ring. For example, —N(Rh)₂ is meant to include, but not belimited to, 1-pyrrolidinyl and 4-morpholinyl. Any one or more of thehydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl,aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkylare optionally substituted by one or more substituents whichindependently are alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy,halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl,—OR^(i), —SR^(i), —OC(O)R^(i), —N(R^(i))₂, —C(O)R^(i), —C(O)OR^(i),—OC(O)N(R^(i))₂, —C(O)N(R^(i))₂, —N(R^(i))C(O)OR, —N(R^(i))C(O)R^(i),—N(R^(i))C(O)N(R^(i))₂, N(R^(i))C(NR^(i))N(R^(i))₂,—N(R^(i))S(O)_(t)R^(i) (where t is 1 or 2), —S(O), or —S(O),N(R^(i))₂(where t is 1 or 2), where each R^(i) is independently hydrogen, alkyl,fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Carbamate” as used herein, refers to a moiety attached to an aminogroup which has the formula —C(O)OR where R is alkyl, fluoroalkyl,carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl. Examples include butare not limited to Boc (tert-butyl-OC(O)—), CBz (benzyl-OC(O)—), Teoc(Me₃SiCH₂CH₂OC(O)—), alloc (allyl-OC(O)—), or Fmoc(9-fluorenylmethyl-OC(O)—) group.

“Substituted silyl” as used herein, refers to a moiety which has theformula R₃Si—. Examples include, but are not limited to, TBDMS(tert-butyldimethylsilyl), TBDPS (tert-butyldiphenylsilyl) or TMS(trimethylsilyl) group.

The term “thiol” refers to —SH groups, and include substituted thiolgroups i.e. —SR^(J) groups, wherein R^(J) are each independently asubstituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, arylaralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

The first aspect of the invention relates to a chiral reagent or a saltthereof. The chiral reagent has following chemical formula (I). The term“chiral reagent” is a chemical composition which is used to producestereocontrolled phosphorus atom-modified nucleotide or oligonucleotidederivatives. The chiral reagent reacts with a nucleotide to form achiral intermediate.

In the formula (I), G¹ and G² are independently a hydrogen atom, a nitrogroup, a halogen atom, a cyano group (—CN), a group of formula (II),(III) or (V), or both G¹ and G² taken together to form a group offormula (IV).

In the formula (II), G²¹ to G²³ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group or C₁₋₃ alkyl group.Preferred examples of G²¹ to G²³ are a hydrogen atom.

In the formula (III), G³¹ to G³³ are independently C₁₋₄ alkyl group,C₆₋₁₄ aryl group C₁₋₄ alkoxy group, C₇₋₁₄ aralkyl group, C₁₋₄ alkylC₆₋₁₄ aryl group, C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkylgroup. Examples of C₁₋₄ alkyl C₆₋₁₄ aryl group are methylphenyl group,and ethylphenyl group. Examples of C₁₋₄ alkoxy C₆₋₁₄ aryl group are amethoxyphenyl group and an ethoxyphenyl group. Examples of C₆₋₁₄ arylC₁₋₄ alkyl groups are a benzyl group and a phenylethyl group. Preferredexamples of G″ to G³³ are independently a methyl group and a phenylgroup.

In the formula (IV), G⁴¹ to G⁴⁶ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group or C₁₋₃ alkyl group.Preferred examples of G⁴¹ to G⁴⁶ are a hydrogen atom.

In the formula (V), G⁵¹ to G⁵³ are independently a hydrogen atom, anitro group, a halogen atom, a cyano group, C₁₋₃ alkyl group or C₁₋₃alkyloxy group.

G³ and G⁴ are independently a hydrogen atom, C₁₋₃ alkyl group, C₆₋₁₄aryl group, or both G³ and G⁴ taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms. Preferredexamples of G³ and G⁴ are that taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms with NH moietyin the formula (I).

A preferred embodiment is that the chiral reagent has following chemicalformula (I′).

In the formula (I′), G¹ and G² are same as above and G¹ and G² areindependently a hydrogen atom, a nitro group, a halogen atom, a cyanogroup, a group of formula (II) or (III), or both G¹ and G² takentogether to form a group of formula (IV).

A preferred embodiment is that the chiral reagent has chemical formula(I′) and each of G¹ and G² is a group of formula (II), wherein G²¹ toG²³ are independently a hydrogen atom, a nitro group, a halogen atom, acyano group or C₁₋₃ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and each of G¹ and G² is a group of formula (II) and each of G²¹ toG²³ is a hydrogen atom.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (II), and G²¹to G²³ are independently a hydrogen atom, a nitro group, a halogen atom,a cyano group or C₁₋₃ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (II), each ofG²¹ and G²² is a hydrogen atom and G²³ is a nitro group (—NO₂).

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom and G² is a group of formula (III), andG³¹ to G³³ are independently C₁₋₄ alkyl group, C₆₋₁₄ aryl group, C₇₋₁₄aralkyl group, C₁₋₄ alkyl C₆₋₁₄ aryl group, C₁₋₄ alkoxy C₆₋₁₄ arylgroup, or C₆₋₁₄ aryl C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom and G² is a group of formula (III), andG³¹ to G³³ are independently C₁₋₄ alkyl group, C₆ aryl group, C₇₋₁₀aralkyl group, C₁₋₄ alkyl C₆ aryl group, C₁₋₄ alkoxy C₆ aryl group, orC₆ aryl C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (III), and G³¹to G³³ are independently C₁₋₄ alkyl group or C₆ aryl group (a phenylgroup). Examples of C₁₋₄ alkyl group are methyl group, ethyl group,n-propyl group, iso-propyl group, n-butyl group and tert-butyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (III), and G³¹to G³³ are independently C₁₋₄ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom, G² is a group of formula (III), and G³¹and G³³ are C₆ aryl group (a phenyl group) and G³² is C₁₋₂ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ and G² taken together to form a group of formula (IV), andG⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, a halogenatom, a cyano group or C₁₋₃ alkyl group.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ and G² taken together to form a group of formula (IV),wherein each of G⁴¹ to G⁴⁶ is a hydrogen atom.

A preferred embodiment is that the chiral reagent has chemical formula(I′) and G¹ is a hydrogen atom and G² is a group of formula (V). Furthereach of G⁵¹ to G⁵³ is independently a hydrogen atom, a nitro group, amethyl group, or a methoxy group. More preferred embodiment is that G¹is a hydrogen atom and G² is a group of formula (V), wherein each of G″and G⁵³ is a hydrogen atom and G⁵³ is a 4-methyl group.

A preferred embodiment is that the chiral reagent is selected from oneof III-a, III-b, V-a, VH-a, VH-b, IX-a, IX-b, XI-a, XIII-a and XIII-b:

-   (S)-2-(Methyldiphenylsilyl)-1-((S)-pyrrolidin-2-yl)ethanol (III-a)-   (R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol (III-b)-   (S)-2-(Trimethylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (V-a)-   (R)-2,2-Diphenyl-1-((S)-pyrrolidin-2-yl)ethanol (VII-a)-   (S)-2,2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (VII-b)-   (R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (IX-a)-   (S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (IX-b)-   (R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (XI-a)-   (S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (XIII-a)-   (R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (XIII-b)

The chiral reagent reacts with a nucleic acid or modified nucleic acidto be an asymmetric auxiliary group. A nucleoside 3′-phosphoramiditederivative, which is an intermediate of manufacturing a stereocontrolledphosphorus atom-modified oligonucleotide derivative, is obtained bychiral reagent reacting with a nucleic acid or modified nucleic acid.

The second aspect of the invention relates to a nucleoside3′-phosphoramidite derivative which is represented by formula (Va) or(Vb). The compounds of formula (Va) and (Vb) are known as monomers thatare used in synthesizing ologonucleotide derivatives. These compoundsare also known as oxazaphospholidine monomers. The sugar moieties of thecompounds represented by formula (Vb) are known as BNA and LNA (when R³is a methylene group).

In the formula (Va) and (Vb), G¹ to G⁴ are same as above, G⁵ is aprotective group of the hydroxyl group, and Bs is a group selected fromthe groups represented by formula (VI) to (XI) or derivatives thereof.

Examples of Bs are an adenine, a thymine, a cytosine, a guanine, anuracil, a 5-methylcytosine, or derivative thereof.

R² is hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen, alkyl, alkenyl,alkynyl, alkyl-Y¹, alkenyl-Y¹, alkynyl-Y¹, aryl-Y¹, heteroaryl-Y¹,—OR^(b), or —SR^(b), wherein R^(b) is a blocking moiety.

Y¹ is O, NR^(d), S, or Se.

R^(d) is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,substituted silyl, carbamate, —P(O)(R^(e))₂, or —HP(O)(R^(e)).

Re is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y²—,alkenyl-Y²-, alkynyl-Y²—, aryl-Y²—, or heteroaryl-Y²—, or a cation whichis Na⁺, Li⁺ or K⁺.

Y² is O, NR^(d), or S.

Preferred examples of alkyl are C₁₋₁₀ alkyl group, preferred examples ofalkenyl are C₂₋₁₀ alkenyl, preferred examples of alkynyl are C₂₋₁₀alkynyl, preferred examples of aryl are C₆₋₁₄ aryl, and preferredexamples of heteroaryl are C₆₋₁₄ heteroaryl.

R³ is a group represented by —CH₂—, —(CH₂)₂—, —CH₂NH—, or —CH₂N(CH₃)—.

Examples of G⁵ the trityl, 4-monomethoxytrityl, 4,4′-dimethoxytrityl,4,4′,4″-trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX).

Bs is an adenine, a thymine, a cytosine, a guanine, or derivativethereof. Bs is a nucleobase or a modified nucleobase. The examples ofthe derivatives are that disclosed in JP 2005-89441 A and arerepresented as follows.

In the above formula, each of R⁸ to R¹⁰ is independently C₁₋₁₀ alkyl,C₆-C₁₀ aryl, C₆-C₁₀ aralkyl, or C₆-C₁₀aryloxyalkyl. Preferred examplesof R⁸ are methyl, isopropyl, phenyl, benzyl, and phenoxymethyl.Preferred examples of R⁹ and R¹⁰ are C₁₋₄ alkyl group.

A preferred embodiment of the second aspect is that the nucleoside3′-phosphoramidite derivative is represented by formula (Va′) or (Vb′).

In the formula (Va′) and (Vb′), 0, G², G⁵, Bs, R², and R³ are same asabove. The nucleoside 3′-phosphoramidite derivative is a chiral monomerwhich is used to produce stereocontrolled phosphorus atom-modifiednucleotides and oligonucleotide derivatives.

Preferred examples of the nucleoside 3′-phosphoramidite derivatives arerepresented by the formula 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a,6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11a, 11b 12a, 12b, 13a, 13b, 14a,14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a,21b, 22a, 22b, 23a, 23b, or 24a. These formulas are described at theExperimental section.

DMTr represents a 4,4′-dimethoxytrityl group and TOM represents atriisopropylsiloxymethyl group.

The examples of using the nucleoside 3′-phosphoramidite derivative aredisclosed in, e.g., JP 2005-89441 A. By repeating steps of condensationand de-protection, it is possible to lengthen the chain ofoligonucleotide derivatives as disclosed therein.

Formula of such an oligonucleotide derivative is shown in formula (X).

In the formula (X), X represents sulfide (═S), C₁₋₃ alkyl, C₁₋₃ alkoxy,C₁₋₃ alkylthio, C₆-C₁₀ aryl, C₆-C₁₀ aralkyl, or C₆-C₁₀ aryloxialkyl.Preferably, X represents sulfide (═S). “n” is an integer that represents1 to 150, 1 to 100, 1 to 50, or 1 to 30. “n” may be preferably 2 to 100,preferably 10 to 100, preferably 10 to 50, and more preferably 15 to 30.

The third aspect of the invention relates to a method for synthesis of astereocontrolled phosphorus atom-modified oligonucleotide derivative.First step is a step of reacting a molecule comprising an achiralH-phosphonate moiety, the first activating reagent and a chiral reagentor a salt thereof to form a monomer. The chiral reagent has chemicalformula (I) or (I′) and the monomer may be represented by formula (Va),(Vb), (Va′), or (Vb′). The monomer reacts with the second activatingreagent and a nucleoside to form a condensed intermediate. Next step isa step of converting the condensed intermediate to the nucleic acidcomprising a chiral X-phosphonate moiety. The method basically based ondisclosure of WO 2010/064146 pamphlet. Namely, fundamental steps aredisclosed as route A and route B therein. In the method the chiralreagent of the present invention is used.

First Scheme relates to synthesis of Chiral Oligos.

Activation Step

An achiral H-phosphonate moiety is treated with the first activatingreagent to form the first intermediate. In one embodiment, the firstactivating reagent is added to the reaction mixture during thecondensation step. Use of the first activating reagent is dependent onreaction conditions such as solvents that are used for the reaction.Examples of the first activating reagent are phosgene, trichloromethylchloroformate, bis(trichloromethyl)carbonate (BTC), oxalyl chloride,Ph₃PCl₂, (PhO)₃PCl₂, N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BopCl),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP).

The example of achiral H-phosphonate moiety is a compound shown in theabove Scheme. DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene. H+DBUmay be, for example, ammonium ion, alkylammonium ion, heteroaromaticiminium ion, or heterocyclic iminium ion, any of which is primary,secondary, tertiary or quaternary, or a monovalent metal ion.

Reacting with Chiral Reagent

After the first activation step, the activated achiral H-phosphonatemoiety reacts with a chiral reagent, which is represented by formula (I)or (I′), to form a chiral intermediate of formula (Va), (Vb), (Va′), or(Vb′).

Stereospecific Condensation Step

A chiral intermediate of Formula Va ((Vb), (Va′), or (Vb′)) is treatedwith the second activating reagent and a nucleoside to form a condensedintermediate. The nucleoside may be solidified. Examples of the secondactivating reagent are 4,5-dicyanoimidazole (DCI),4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole(NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole(BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate(CMPT), N-cyanomethylpiperidinium triflate,N-cyanomethyldimethylammonium triflate. A chiral intermediate of FormulaVa ((Vb), (Va′), or (Vb′)) may be isolated as a monomer. Usually, thechiral intermediate of Va ((Vb), (Va′), or (Vb′)) is not isolated andundergoes a reaction in the same pot with a nucleoside or modifiednucleoside to provide a chiral phosphite compound, a condensedintermediate. In other embodiments, when the method is performed viasolid phase synthesis, the solid support comprising the compound isfiltered away from side products, impurities, and/or reagents.

Capping Step

If the final nucleic acid is larger than a dimer, the unreacted—OHmoiety is capped with a blocking group and the chiral auxiliary in thecompound may also be capped with a blocking group to form a cappedcondensed intermediate. If the final nucleic acid is a dimer, then thecapping step is not necessary.

Modifying Step

The compound is modified by reaction with an electrophile. The cappedcondensed intermediate may be executed modifying step. In someembodiments of the method, the modifying step is performed using asulfur electrophile, a selenium electrophile or a boronating agent. Thepreferred examples of modifying steps are step of oxidation andsulfurization.

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas:

S₈ (Formula B), Z¹—S—S—Z², or Z¹—S—V—Z².

Z¹ and Z² are

-   -   independently alkyl, aminoalkyl, cycloalkyl, heterocyclic,        cycloalkylalkyl, heterocycloalkyl,    -   aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide,        imide, or thiocarbonyl, or Z¹    -   and Z² are taken together to form a 3 to 8 membered alicyclic or    -   heterocyclic ring, which may be substituted or unsubstituted; V        is SO₂, O, or NR^(f); and R^(f) is hydrogen, alkyl, alkenyl,        alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof following Formula A, B, C, D, E, or F:

In some embodiments of the method, the selenium electrophile is acompound having one of the following formulas:

Se (Formula G), Z³—Se—Se—Z⁴, or Z³—Se—V—Z⁴

Z³ and Z⁴ are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic,cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,heteroaryloxy, acyl, amide, imide, or thiocarbonyl, or Z³ and Z⁴ aretaken together to form a 3 to 8 membered alicyclic or heterocyclic ring,which may be substituted or unsubstituted; V is SO₂, S, O, or NR^(f);and R^(f) is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

In some embodiments of the method, the selenium electrophile is acompound of Formula G, H, I, J, K, or L.

In some embodiments of the method, the boronating agent isborane-N,N-diisopropylethylamine (BH₃ DIPEA), borane-pyridine (BH₃ Py),borane-2-chloropyridine (BH₃ CPy), borane-aniline (BH₃ An),borane-tetrahydrofiirane (BH₃ THF), or borane-dimethylsulfide (BH₃Me₂S).

In some embodiments of the method, the modifying step is oxidation step.Oxidation step is disclosed in, e.g., JP 2010-265304 A andWO2010/064146.

Chain Elongation Cycle and De-protection Step

The capped condensed intermediate is deblocked to remove the blockinggroup at the 5′-end of the growing nucleic acid chain to provide acompound. The compound is optionally allowed to re-enter the chainelongation cycle to form a condensed intermediate, a capped condensedintermediate, a modified capped condensed intermediate, and a5′-deprotected modified capped intermediate. Following at least oneround of chain elongation cycle, the 5′-deprotected modified cappedintermediate is further deblocked by removal of the chiral auxiliaryligand and other protecting groups, e.g., nucleobase, modifiednucleobase, sugar and modified sugar protecting groups, to provide anucleic acid. In other embodiments, the nucleoside comprising a 5′-OHmoiety is an intermediate from a previous chain elongation cycle asdescribed herein. In yet other embodiments, the nucleoside comprising a5′-OH moiety is an intermediate obtained from another known nucleic acidsynthetic method. In embodiments where a solid support is used, thephosphorus-atom modified nucleic acid is then cleaved from the solidsupport. In certain embodiments, the nucleic acids is left attached onthe solid support for purification purposes and then cleaved from thesolid support following purification.

Based on the present method, it is possible to use stable andcommercially available materials as starting materials. It is possibleto produce stereocontrolled phosphorus atom-modified oligonucleotidederivatives using an achiral starting material.

As shown in a working example, the method of the present invention doesnot cause degradations under the de-protection steps. Further the methoddoes not require special capping agents to produce phosphorusatom-modified oligonucleotide derivatives.

The fourth aspect of the invention relates to a method for the synthesisof stereocontrolled phosphorus atom-modified oligonucleotide derivativesusing a chiral monomer. The first step is reacting a nucleoside3′-phosphoramidite derivative which is represented by formula (Va),(Vb), (Va′), or (Vb′) with the second activating reagent and anucleoside to form a condensed intermediate. The second step isconverting the condensed intermediate to the nucleic acid comprising achiral X-phosphonate moiety.

Second Scheme relates to synthesis of Chiral Oligos using a monomer ofFormula Va ((Vb), (Va′), or (Vb′)). The second Scheme based on themethod disclosed in JP 2005-89441 A.

The detailed conditions of the above scheme are similar to that of thefirst scheme. The starting material of formula Va (Vb), especially offormula Va′ (or Vb′), is chemically stable. As shown in a workingexample, the method of the present invention does not cause degradationsunder the de-protection steps. Further the method does not requirespecial capping agents to produce phosphorus atom-modifiedoligonucleotide derivatives.

Mechanism for the removal of auxiliaries is shown as follows:

In the above scheme, Nu stands for Nucleophile. The above mechanism isthought to be different from the previous mechanism for the removal ofauxiliaries.

EXAMPLES Abbreviation

-   -   ac: acetyl    -   bz: benzoyl    -   CSO: (1S)-(+)-(10-camphorsulfonyl)oxaziridine    -   DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCA: dichloroacetic acid    -   DCM: dichloromethane, CH₂Cl₂    -   DMTr: 4,4′-dimethoxytrityl    -   Tr: trityl, triphenylmethyl    -   MeIm: N-methylimidazole    -   NIS: N-iodosuccinimide    -   pac: phenoxyacetyl    -   Ph: phenyl    -   PhIMT: N-phenylimidazolium triflate    -   POS: 3-phenyl-1,2,4-dithiazoline-5-one    -   TBS: tert-butyldimethylsilyl    -   TBDPS: tert-butyldiphenylsilyl    -   TOM: triisopropylsiloxymethyl    -   TFA: trifluoroacetic acid

Example 1 (S)-1-Tritylpyrrolidin-2-carbaldehyde (I-a)

Compound I-a was synthesized from L-proline according to the proceduredescribed in the literature (Guga, P. Curr. Top. Med. Chem. 2007, 7,695-713.).

Example 2 (R)-1-Tritylpyrrolidin-2-carbaldehyde (I-b)

Compound I-b was synthesized from D-proline in a similar manner tocompound I-a.

Example 3(S)-2-(Methyldiphenylsilyl)-1-((S)-1-tritylpyrrolidin-2-yl)ethanol(II-a)

To a solution of methyldiphenylsilylmethyl magnesium chloride in THFprepared from chloromethyldiphenylmethylsilane (4.02 g, 16.3 mmol) andmagnesium (402 mg, 16.3 mmol) in THF (14 mL) was added I-a (2.79 g, 8.14mmol) in THF (30 mL) solution with ice cooling. After stirring for 1.5 hwith ice cooling, the mixture warmed to room temperature and continuedstirring for 30 min. Saturated aqueous NH₄Cl (100 mL) was added to thereaction mixture at 0 degrees C., and extraction was performed withdiethylether (100 mL) for three times. The combined extract was driedover Na_(z) SO₄, filtered and concentrated under reduced pressure. Theresidue was chromatographed on silica gel afforded II-a as a colorlessfoam (3.91 g, 87%).

¹H NMR (300 MHz, CDCl₃) d 7.48-7.08 (25H, m), 4.33-4.23 (1H, m),3.16-2.89 (3H, m), 2.84 (1H, brs), 1.70-1.54 (1H, m), 1.35 (1H, dd,J=14.7, 6.3 Hz), 1.10 (1H, dd, J=14.7, 8.1 Hz), 1.18-1.05 (1H, m),1.04-0.90 (1H, m), 0.34 (3H, s), −0.17-−0.36 (1H, m).

Example 4 (S)-2-(Methyldiphenylsilyl)-1-((S)-pyrrolidin-2-yl)ethanol(III-a)

II-a (3.91 g, 7.06 mmol) was dissolved in 3% DCA in DCM (70 mL), andstirred for 10 min at room temperature. To the mixture, 1M NaOH (200 mL)was added, and extraction was performed with DCM (100 mL) for threetimes. The combined extract was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was chromatographed onsilica gel afforded III-a as a light yellow oil (1.99 g, 90%).

¹H NMR (300 MHz, CDCl₃) d 7.57-7.52 (5H, m), 7.38-7.33 (5H, m), 3.77(1H, ddd, J=8.9, 5.4, 3.5 Hz), 3.01 (1H, dt, J=7.4, 3.6 Hz), 2.97-2.79(2H, m), 2.27 (2H, brs), 1.76-1.53 (4H, m), 1.38 (1H, dd, J=15.0, 9.0Hz), 1.24 (1H, dd, J=15.0, 5.4 Hz), 0.65 (3H, s); ¹³C NMR (100.4 MHz,CDCl₃) d 137.4, 137.1, 134.6, 134.5, 129.1, 127.8, 69.5, 64.1, 47.0,25.8, 24.0, 19.6, −3.4. MALDI TOF-MS m/z Calcd for C₉H₂₆NOSi [M+H]⁺312.18. found 312.06.

Example 5(R)-2-(Methyldiphenylsilyl)-1-((R)-1-tritylpyrrolidin-2-yl)ethanol(II-b)

Compound II-b was obtained by using I-b instead of I-a in a similarmanner to compound II-a.

¹H NMR (300 MHz, CDCl₃) d 7.48-7.12 (25H, m), 4.33-4.24 (1H, m),3.16-2.89 (3H, m), 2.86 (1H, brs), 1.69-1.52 (1H, m), 1.35 (1H, dd,J=14.4, 6.0 Hz), 1.10 (1H, dd, J=14.4, 8.4 Hz), 1.18-1.05 (1H, m),1.03-0.89 (1H, m), 0.33 (3H, s), −0.19-−0.39 (1H, m);

¹³C NMR (75.5 MHz, CDCl₃) d 144.5, 137.5, 136.8, 134.6, 134.3, 129.8,129.0, 127.8, 127.7, 127.4, 126.1, 77.9, 71.7, 65.1, 53.5, 25.0, 24.8,19.6, −4.0. MALDI TOF-MS m/z Calcd for C₃₈H₄₀NOSi [M+H]⁺ 554.29. found554.09.

Example 6 (R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol(111-b)

Compound III-b was obtained by using II-b instead of II-a in a similarmanner to compound III-a.

¹H NMR (300 MHz, CDCl₃) d 7.58-7.52 (5H, m), 7.38-7.33 (5H, m), 3.78(1H, ddd, J=9.0, 5.1, 3.6 Hz), 3.00 (1H, dt, J=7.4, 3.3 Hz), 2.97-2.78(2H, m), 2.19 (2H, brs), 1.76-1.53 (4H, m), 1.38 (1H, dd, J=14.6, 9.0Hz), 1.24 (1H, dd, J=14.6, 5.1 Hz), 0.66 (3H, s); ¹³C NMR (75.5 MHz,CDCl₃) d 137.5, 137.1, 134.5, 134.4, 129.0, 127.7, 69.2, 64.2, 46.9,25.8, 24.0, 19.7, −3.4. MALDI TOF-MS m/z Calcd for C₁₉H₂₆NOSi [M+H]⁺312.18. found 312.09.

Example 7 (S)-2-(Trimethylsilyl)-1-((S)-1-tritylpyrrolidin-2-yl)ethanol(IV-a)

Compound IV-a was obtained by using “chloromethyltrimethylsilane”instead of “chloromethyldiphenylmethylsilane” in a similar manner tocompound II-a.

¹H NMR (300 MHz, CDCl₃) d 7.58-7.51 (5H, m), 7.31-7.14 (10H, m), 4.13(1H, dt, J=7.5, 3.0 Hz), 3.39-3.31 (1H, m), 3.20-2.99 (2H, m), 2.84 (1H,s), 1.74-1.57 (1H, m), 1.29-1.10 (2H, m), 0.74 (1H, dd, J=14.4, 7.2 Hz),0.46 (1H, dd, J=14.4, 7.2 Hz), −0.15 (9H, s). MALDI TOF-MS m/z Calcd forC₂₈H₃₆NOSi [M+H]⁺ 430.26. found 430.09.

Example 8 (S)-2-(Trimethylsilyl)-1-pyrrolidin-2-yl)ethanol (V-a)

Compound V-a was obtained by using IV-a instead of II-a in a similarmanner to compound III-a.

¹H NMR (300 MHz, CDCl₃) d 3.76 (1H, ddd, J=8.8, 5.7, 3.3 Hz), 3.08 (1H,dt, J=7.8, 3.3 Hz), 3.02-2.87 (2H, m), 2.48 (2H, brs), 1.81-1.58 (4H,m), 0.83 (1H, dd, J=14.7, 8.7 Hz), 0.68 (1H, dd, J=14.7, 6.0 Hz), 0.05(9H, s); ¹³C NMR (75.5 MHz, CDCl₃) d 69.6, 64.3, 46.9, 25.8, 23.9, 22.0,−0.8. MALDI TOF-MS m/z Calcd for C₉H₂₂NOSi [M+H]⁺ 188.15. found 188.00.

Example 9 (R)-2,2-Diphenyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (VI-a)

To a solution of diphenylmethane (6.7 mL, 40 mmol) in anhydrous THF (36mL), n-BuLi (1.67M solution of Hexane, 24 mL, 40 mmol) was addeddropwise at room temperature and stirred for 1 h. To the mixture, I-a(3.41 g, 10 mmol), which was dried by repeated coevaporations withtoluene, in anhydrous THF (40 mL) was slowly added at 0 degrees C., andcontinued stirring for 45 min. A saturated NH₄Cl aqueous solution (100mL) and Et₂O (100 mL) were then added, and the organic layer wasseparated and the aqueous layer was extracted with Et₂O (2 x 100 mL).The organic layer were combined, dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified bychromatography on silica gel to afford VI-a (1.41 g, 28%) as white foam.

¹H NMR (300 MHz, CDCl₃) d 7.45-7.01 (23H, m), 6.67-6.61 (2H, m), 4.80(1H, d, J=10.8 Hz), 3.63 (1H, d, J=10.8 Hz), 3.36-3.27 (1H, m),3.23-3.09 (1H, m), 3.02-2.89 (1H, m), 2.66 (1H, s), 1.90-1.75 (1H, m),1.32-1.04 (2H, m), 0-−0.18 (1H, m).

Example 10 (R)-2,2-Diphenyl-1((S)-pyrrolidin-2-yl)ethanol (VII-a)

Compound VII-a was obtained by using VI-a instead of II-a in a similarmanner to compound III-a.

¹H NMR (300 MHz, CDCl₃) d 7.44-7.38 (2H, m), 7.33-7.14 (8H, m), 4.46(1H, dd, J=9.9, 3.3 Hz), 3.91 (1H, d, J=9.9 Hz), 3.02-2.88 (2H, m),2.81-2.69 (1H, m), 2.52 (2H, brs), 1.88-1.56 (4H, m); ¹³C NMR (75.5 MHz,CDCl₃) d 142.3, 142.0, 128.6, 128.5, 128.4, 128.2, 126.5, 126.4, 73.5,60.1, 55.8, 46.6, 25.8, 23.4. MALDI TOF-MS m/z Calcd for C₁₈H₂₂NO [M+H]⁺268.17. found 268.06.

Example 11 (S)-2.2-Diphenyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol(VI-b)

Compound VI-b was obtained by using I-b instead of I-a in a similarmanner to compound VI-a.

¹H NMR (300 MHz, CDCl₃) d 7.44-7.37 (6H, m), 7.30-7.01 (17H, m),6.66-6.61 (2H, m), 4.80 (1H, d, J=10.8 Hz), 3.63 (1H, d, J=10.8 Hz),3.36-3.28 (1H, m), 3.22-3.09 (1H, m), 3.01-2.89 (1H, m), 2.66 (1H, s),1.90-1.75 (1H, m), 1.29-1.04 (2H, m), 0.00-0.19 (1H, m); ¹³C NMR (75.5MHz, CDCl₃) d 144.2, 142.9, 141.6, 130.0, 128.5, 128.4, 127.9, 127.8,127.4, 126.4, 126.2, 77.9, 75.9, 61.9, 55.4, 53.4, 24.7, 24.5. MALDITOF-MS m/z Calcd for C₃₇H₃₆NO [M+H]⁺ 510.28. found 510.11.

Example 12 (S1-2.2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (VII-b)

Compound VII-b was obtained by using VI-b instead of VI-a in a similarmanner to compound VII-a.

¹H NMR (300 MHz, CDCl₃) d 7.45-7.14 (10H, m), 4.45 (1H, dd, J=9.9, 3.3Hz), 3.91 (1H, d, J=9.9 Hz), 3.00-2.89 (2H, m), 2.82-2.71 (1H, m), 2.40(2H, brs), 1.87-1.55 (4H, m); ¹³C NMR (75.5 MHz, CDCl₃) d 142.3, 142.0,128.5, 128.3, 128.1, 126.3, 126.2, 73.4, 60.1, 55.9, 46.5, 25.8, 23.5.MALDI TOF-MS m/z Calcd for C₁₈H₂₂NO [M+H]⁺ 268.17. found 268.03.

Example 13 (R)-2-(4-Nitrophenyl)-1-((S)-1-tritylpyrrolidin-2-yl)ethanol(VIII-a)

Compound VIII-a was obtained by using “4-nitrobenzylchloride” instead of“diphenylmethane” in a similar manner to compound VI-a.

¹H NMR (300 MHz, CDCl₃) d 8.09-8.03 (2H, m), 7.49-7.43 (6H, m),7.28-7.09 (11H, m), 4.23 (1H, ddd, J=8.3, 5.6, 3.0 Hz), 3.43-3.33 (1H,m), 3.23-3.11 (1H, m), 3.07-2.96 (1H, m), 2.83 (1H, brs), 2.74 (1H, dd,J=13.8, 8.4 Hz), 2.49 (1H, dd, J=13.8, 5.1 Hz), 1.83-1.67 (1H, m),1.41-1.17 (2H, m), 0.27-0.08 (1H, m); ¹³C NMR (75.5 MHz, CDCl₃) d 147.3,146.3, 144.3, 129.8, 129.6, 127.5, 126.3, 123.4, 77.9, 74.8, 63.5, 53.2,39.5, 25.0, 24.9. MALDI TOF-MS m/z Calcd for C₃₁H₃₁N₂O₃ [M+H]⁺ 479.23.found 479.08.

Example 14 (R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (IX-a)

Compound IX-a was obtained by using VIII-a instead of VI-a in a similarmanner to compound VII-a.

¹H NMR (300 MHz, CDCl₃) d 8.15 (2H, d, J=8.7 Hz), 7.42 (2H, d, J=8.7Hz), 3.86-3.79 (1H, m), 3.16-3.07 (1H, m), 2.99-2.68 (6H, m), 1.84-1.68(4H, m); ¹³C NMR (75.5 MHz, CDCl₃) d 147.4, 146.2, 129.9, 123.2, 72.4,62.0, 46.6, 40.4, 25.7, 24.4. MALDI TOF-MS m/z Calcd for C₁₂H₁₇N₂O₃[M+H]⁺ 237.12. found 237.01.

Example 15 (S)-2-(4-Nitrophenyl)-1-((R)-1-tritylpyrrolidin-2-yl)ethanol(VIII-b)

Compound VIII-b was obtained by using I-b instead of I-a in a similarmanner to compound VIII-a.

¹H NMR (300 MHz, CDCl₃) d 8.09-8.04 (2H, m), 7.49-7.43 (6H, m),7.28-7.09 (11H, m), 4.22 (1H, ddd, J=8.4, 5.6, 3.0 Hz), 3.43-3.33 (1H,m), 3.24-3.10 (1H, m), 3.08-2.94 (1H, m), 2.81 (1H, brs), 2.75 (1H, dd,J=14.0, 8.1 Hz), 2.49 (1H, dd, J=14.0, 5.1 Hz), 1.81-1.67 (1H, m),1.40-1.16 (2H, m), 0.26-0.09 (1H, m); ¹³C NMR (75.5 MHz, CDCl₃) d 147.3,144.3, 129.8, 129.6, 129.4, 126.3, 123.5, 77.9, 74.8, 63.5, 53.2, 39.5,25.0, 24.9. MALDI TOF-MS m/z Calcd for C₃₁H₃₁N₂O₃ [M+H]⁺ 479.23. found479.08.

Example 16 (S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (IX-b)

Compound IX-b was obtained by using VIII-b instead of VIII-a in asimilar manner to compound IX-a.

¹H NMR (300 MHz, CDCl₃) d 8.19-8.13 (2H, m), 7.45-7.39 (2H, m), 3.83(1H, ddd, J=7.7, 5.4, 3.9 Hz), 3.14 (1H, dt, J=7.7, 3.9 Hz), 3.01-2.87(2H, m), 2.83 (1H, d, J=3.3 Hz), 2.81 (1H, s), 2.62 (2H, brs), 1.79-1.72(4H, m); ¹³C NMR (75.5 MHz, CDCl₃) d 147.3, 146.5, 130.0, 123.5, 72.7,61.7, 46.7, 40.1, 25.8, 24.2. MALDI TOF-MS m/z Calcd for C₁₂H₁₇N₂O₃[M+H]⁺ 237.12. found 237.02.

Example 17 (R)-(9H-Fluoren-9-yl)((S)-1-tritylpyrrolidin-2-yl)methanol(X-a)

Compound X-a was obtained by using “fluorene” instead of“diphenylmethane” in a similar manner to compound VI-a.

¹H NMR (300 MHz, CDCl₃) d 7.70 (1H, d, J=7.5 Hz), 7.66 (1H, d, J=7.8Hz), 7.55 (2H, d, J=7.5 Hz), 7.44-7.09 (18H, m), 6.87-6.62 (1H, m),4.55-4.48 (1H, m), 4.06 (1H, d, J=7.5 Hz), 3.43-3.34 (1H, m), 3.18-3.06(1H, m), 2.98-2.88 (1H, m), 2.85 (1H, brs), 1.42-1.24 (1H, m), 1.18-1.04(1H, m), 0.53-0.39 (1H, m), −0.02-−0.20 (1H, m); MALDI TOF-MS m/z Calcdfor C₃₇H₃₄NO [M+H]⁺ 508.26. found 508.12.

Example 18 (R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (XI-a)

Compound XI-a was obtained by using X-a instead of II-a in a similarmanner to compound III-a.

¹H NMR (300 MHz, CDCl₃) d 7.76 (2H, d, J=7.5 Hz), 7.68 (2H, t, J=8.0Hz), 7.43-7.35 (2H, m), 7.34-7.25 (2H, m), 4.28 (1H, d, J=6.3 Hz), 4.03(1H, dd, J=6.5, 4.2 Hz), 3.19-3.11 (1H, m), 2.97-2.88 (1H, m), 2.86-2.76(1H, m), 2.02 (2H, brs), 1.77-1.53 (3H, m), 1.38-1.23 (1H, m); MALDITOF-MS m/z Calcd for C₁₈H₂₀NO [M+H]⁺ 266.15. found 266.04.

Example 19 (S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (XII-a)

Compound XII-a was obtained by using “chloromethyl p-tolyl sulfone”instead of “chloromethyldiphenylmethylsilane” in a similar manner tocompound II-a.

¹H NMR (600 MHz, CDCl₃) d 7.66 (2H, d, J=8.4 Hz), 7.48-7.44 (6H, m),7.35 (2H, d, J=7.2 Hz), 7.21-7.13 (9H, m), 4.39-4.36 (1H, m), 3.33 (1H,s), 3.24-3.20 (1H, m), 3.19-3.10 (2H, m), 2.98-2.92 (2H, m), 2.49 (3H,s), 1.55-1.49 (1H, m), 1.33-1.26 (1H, m), 1.12-1.04 (1H, m), 0.22-0.14(1H, m); ¹³C NMR (150.9 MHz, CDCl₃) d 144.6, 144.5, 136.3, 129.9, 129.5,128.1, 127.5, 126.2, 78.0, 69.1, 63.9, 60.2, 52.6, 25.5, 24.7, 21.7.

Example 20 (S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (XIII-a)

Compound XIII-a was obtained by using XII-a instead of II-a in a similarmanner to compound III-a.

¹H NMR (600 MHz, CDCl₃) d 7.82 (2H, d, J=8.4 Hz), 7.37 (2H, d, J=8.4Hz), 4.01 (1H, ddd, J=12.0, 5.1, 3.0 Hz), 3.32 (1H, dd, J=14.4, 3.0 Hz),3.25 (1H, dd, J=14.4, 9.0 Hz), 3.16 (1H, dt, J=7.8, 5.1 Hz), 2.90-2.82(2H, m), 2.46 (3H, s), 2.04 (2H, brs), 1.78-1.63 (3H, m), 1.62-1.55 (1H,m); ¹³C NMR (150.9 MHz, CDCl₃) d 144.5, 136.7, 129.7, 127.7, 67.4, 61.8,60.1, 46.7, 25.7, 21.4. MALDI TOF-MS m/z Calcd for C₁₃H₂₀NO₃S [M+H]⁺270.12. found 270.04.

Example 2 (R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (XII-b)

Compound XII-b was obtained by using I-b instead of I-a in a similarmanner to compound XII-a.

¹H NMR (600 MHz, CDCl₃) d 7.66 (2H, d, J=8.4 Hz), 7.47-7.44 (6H, m),7.35 (2H, d, J=7.8 Hz), 7.21-7.13 (9H, m), 4.37 (1H, dt, J=8.6, 2.4 Hz),3.33 (1H, s), 3.23-3.20 (1H, m), 3.19-3.12 (2H, m), 2.98-2.92 (2H, m),2.49 (3H, s), 1.56-1.49 (1H, m), 1.32-1.26 (1H, m), 1.11-1.03 (1H, m),0.23-0.15 (1H, m); ¹³C NMR (150.9 MHz, CDCl₃) d 144.6, 144.5, 136.3,129.9, 129.6, 128.1, 127.6, 126.2, 78.0, 69.1, 63.9, 60.2, 52.6, 25.5,24.7, 21.7.

Example 21 (R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (XIII-b)

Compound XIII-b was obtained by using XII-b instead of XII-a in asimilar manner to compound XIII-a ¹H NMR (600 MHz, CDCl₃) d 7.82 (2H, d,J=8.4 Hz), 7.37 (2H, d, J=8.4 Hz), 4.01 (1H, ddd, J=9.0, 5.1, 3.0 Hz),3.32 (1H, dd, J=14.4, 3.0 Hz), 3.25 (1H, dd, J=14.4, 9.0 Hz), 3.17 (1H,dt, J=7.2, 5.1 Hz), 2.89-2.83 (2H, m), 2.46 (3H, s), 2.04 (2H, brs),1.79-1.64 (3H, m), 1.62-1.55 (1H, m); ¹³C NMR (150.9 MHz, CDCl₃) d144.8, 136.6, 129.8, 127.9, 67.7, 61.8, 60.1, 46.8, 25.9, 25.8, 21.6.MALDI TOF-MS m/z Calcd for C₁₃H₂₀NO₃S [M+H]⁺ 270.12. found 270.05.

Example 22 Oxazaphospholidine monomer 3a

III-a (560 mg, 1.80 mmol) were dried by repeated coevaporations with drytoluene and dissolved in dry diethylether (0.90 mL) under argon.N-Methylmorpholine (400 mL, 3.60 mmol) was added to the solution, andthe resultant solution was added dropwise to a solution of PCl₃ (160 mL,1.80 mmol) in dry diethylether (0.90 mL) at 0 degrees C. under argonwith stirring. The mixture was then allowed to warm to room temperatureand stirred for 30 min. The resultant N-methylmorpholine hydrochloridewas removed by filtration under nitrogen, and the filtrate wasconcentrated to dryness under reduced pressure to afford crude2-chloro-1,3,2-oxazaphospholidine derivative. The crude materials weredissolved in freshly distilled THF (3.6 mL) to make 0.5 M solutions,which were used to synthesize the nucleoside 3′-O-oxazaphospholidineswithout further purification.

5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine (636 mg, 0.84mmol) was dried by repeated coevaporations with dry toluene, anddissolved in freshly distilled THF (2.5 mL) under argon. Et₃N (0.58 mL,4.2 mmol) was added, and the mixture was cooled to −78 degrees C. A 0.5M solution of the corresponding crude 2-chloro-1,3,2-oxazaphospholidinederivative in freshly distilled THF (3.6 mL, 1.80 mmol) was addeddropwise via a syringe, and the mixture was stirred for 15 min at roomtemperature. A saturated NaHCO₃ aqueous solution (70 mL) and CHCl₃ (70mL) were then added, and the organic layer was separated and washed withsaturated NaHCO₃ aqueous solutions (2 x 70 mL). The combined aqueouslayers were back-extracted with CHCl₃ (70 mL). The organic layers werecombined, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was purified by chromatography on silica gel toafford 3a (829 mg, 90%) as a white foam.

¹H NMR (300 MHz, CDCl₃) d 8.77 (1H, brs), 7.99 (1H, s), 7.54-6.98 (24H,m), 6.81-6.73 (4H, m), 6.35 (1H, dd, J=8.0, 6.3 Hz), 4.89-4.73 (4H, m),4.68 (2H, brs), 4.05-3.98 (1H, m), 3.75 (6H, s), 3.62-3.46 (1H, m),3.41-3.20 (3H, m), 3.18-3.04 (1H, m), 3.08 (2H, t, J=6.6 Hz), 2.58-2.36(2H, m), 1.94-1.59 (2H, m), 1.56 (1H, dd, J=15.0, 8.7 Hz), 1.43 (1H, dd,J=15.0, 5.7 Hz), 1.33-1.16 (2H, m), 0.62 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) d 153.5 (1P, s).

Example 23 Oxazaphospholidine monomer 3b

Compound 3b was obtained by using III-b instead of III-a in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.80 (1H, brs), 7.96 (1H, s), 7.54-6.96 (24H,m), 6.79-6.71 (4H, m), 6.19 (1H, t, J=6.6 Hz), 4.90-4.73 (4H, m), 4.66(2H, brs), 4.16-4.08 (1H, m), 3.76 (6H, s), 3.60-3.36 (2H, m), 3.29 (1H,d, J=3.9 Hz), 3.27-3.12 (2H, m), 3.09 (2H, t, J=6.6 Hz), 2.59-2.46 (1H,m), 2.07-1.97 (1H, m), 1.94-1.41 (5H, m), 1.36-1.18 (1H, m), 0.65 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.1 (1P, s).

Example 24 Oxazaphospholidine monomer 1a

Compound 1a was obtained by using “5′-O-(DMTr)-6-N-(benzoyl)adenosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (600 MHz, CDCl₃) d 8.71 (1H, s), 8.12 (1H, s), 8.04 (2H, d, J=7.8Hz), 7.62-7.15 (23H, m), 6.80-6.75 (4H, m), 6.37 (1H, dd, J=7.8, 6.0Hz), 4.94-4.88 (1H, m), 4.80 (1H, ddd, J=12.0, 6.0, 5.4 Hz), 4.07-4.04(1H, m), 3.76 (6H, s), 3.58-3.49 (1H, m), 3.41-3.34 (1H, m), 3.33 (1H,dd, J=10.8, 4.8 Hz), 3.25 (1H, dd, J=10.8, 4.8 Hz), 3.13-3.06 (1H, m),2.66-2.58 (1H, m), 2.40-2.35 (1H, m), 1.91-1.84 (1H, m), 1.73-1.66 (1H,m), 1.56 (1H, dd, J=15.0, 9.0 Hz), 1.44 (1H, dd, J=15.0, 5.4 Hz),1.47-1.41 (1H, m), 1.30-1.23 (1H, m), 0.63 (3H, s); ³¹P NMR (243.0 MHz,CDCl₃) d 151.8 (1P, s).

Example 25 Oxazaphospholidine monomer 1b

Compound 1b was obtained by using III-b instead of III-a in a similarmanner to compound 1a.

¹H NMR (300 MHz, CDCl₃) d 9.06 (1H, brs), 8.76 (1H, s), 8.12 (1H, s),8.07-7.99 (2H, m), 7.64-7.14 (22H, m), 6.83-6.75 (4H, m), 6.25 (1H, t,J=6.6 Hz), 4.86-4.75 (2H, m), 4.20-4.15 (1H, m), 3.77 (6H, s), 3.61-3.38(2H, m), 3.36 (1H, dd, J=10.2, 4.2 Hz), 3.27 (1H, dd, J=10.2, 4.2 Hz),3.27-3.13 (1H, m), 2.71-2.59 (1H, m), 2.12-2.01 (1H, m), 1.94-1.42 (5H,m), 1.36-1.20 (1H, m), 0.67 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.3(1P, s).

Example 26 Oxazaphospholidine monomer 2a

Compound 2a was obtained by using “5′-O-(DMTr)-4-N-(isobutyryl)cytidine”instead of “5′-O— (DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.33 (1H, brs), 8.17 (1H, d, J=7.5 Hz),7.52-7.22 (19H, m), 7.07 (1H, d, J=7.5 Hz), 6.88-6.81 (4H, m), 6.20 (1H,t, J=6.2 Hz), 4.81-4.64 (2H, m), 3.93-3.87 (1H, m), 3.79 (6H, s),3.59-3.43 (1H, m), 3.39-3.29 (3H, m), 3.16-3.02 (1H, m), 2.69-2.52 (2H,m), 2.12-2.00 (1H, m), 1.91-1.50 (3H, m), 1.47-1.32 (2H, m), 1.27-1.16(7H, m), 0.60 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 154.8 (1P, s).

Example 27 Oxazaphospholidine monomer 2b

Compound 2b was obtained by using III-b instead of III-a in a similarmanner to compound 2a.

¹H NMR (300 MHz, CDCl₃) d 8.33 (1H, d, J=7.5 Hz), 8.23 (1H, brs),7.57-7.22 (19H, m), 7.12 (1H, d, J=7.5 Hz), 6.88-6.81 (4H, m), 6.15 (1H,dd, J=6.6, 4.2 Hz), 4.82-4.63 (2H, m), 4.03-3.97 (1H, m), 3.80 (6H, s),3.55-3.26 (4H, m), 3.19-3.05 (1H, m), 2.59 (1H, quintet, J=6.9 Hz),2.39-2.27 (1H, m), 2.21-2.10 (1H, m), 1.90-1.56 (3H, m), 1.50-1.32 (2H,m), 1.26-1.17 (7H, m), 0.66 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.2(1P, s).

Example 28 Oxazaphospholidine monomer 4a

Compound 4a was obtained by using “5′-O-(DMTr)thymidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.58-7.23 (21H, m), 6.86-6.79 (4H, m), 6.35(1H, dd, J=8.1, 5.7 Hz), 4.79-4.67 (2H, m), 3.83-3.78 (1H, m), 3.78 (6H,s), 3.59-3.43 (1H, m), 3.34 (1H, dd, J=10.5, 2.4 Hz), 3.35-3.24 (1H, m),3.20 (1H, dd, J=10.5, 2.4 Hz), 3.16-3.02 (1H, m), 2.36-2.26 (1H, m),2.15-2.02 (1H, m), 1.92-1.77 (1H, m), 1.74-1.59 (1H, m), 1.52 (1H, dd,J=14.7, 9.0 Hz), 1.40 (3H, s), 1.45-1.15 (3H, m), 0.60 (3H, s); ³¹P NMR(121.5 MHz, CDCl₃) d 153.7 (1P, s).

Example 29 Oxazaphospholidine monomer 4b

Compound 4b was obtained by using III-b instead of III-a in a similarmanner to compound 4a.

¹H NMR (300 MHz, CDCl₃) d 8.46 (1H, brs), 7.59-7.20 (20H, m), 6.86-6.79(4H, m), 6.26 (1H, t, J=6.8 Hz), 4.78-4.65 (2H, m), 4.01-3.95 (1H, m),3.78 (6H, s), 3.55-3.40 (1H, m), 3.42 (1H, dd, J=10.5, 2.7 Hz),3.40-3.28 (1H, m), 3.22 (1H, dd, J=10.5, 3.0 Hz), 3.19-3.06 (1H, m),2.16-1.95 (2H, m), 1.90-1.54 (3H, m), 1.49-1.35 (1H, m), 1.43 (3H, s),1.34-1.17 (2H, m), 0.67 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 156.2 (1P,s).

Example 30 Oxazaphospholidine monomer 5a

Compound 5a was obtained by using

“5′-O-(DMTr)-2′-O-methyl-6-N-(benzoyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.66 (1H, s), 8.13 (1H, s), 8.03 (2H, d, J=7.2Hz), 7.64-7.16 (23H, m), 6.79 (4H, d, J=8.7 Hz), 6.08 (1H, d, J=6.3 Hz),4.91-4.81 (1H, m), 4.77-4.69 (1H, m), 4.64-4.57 (1H, m), 4.15-4.10 (1H,m), 3.76 (6H, s), 3.60-3.23 (4H, m), 3.35 (3H, s), 3.14-3.00 (1H, m),1.90-1.19 (6H, m), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 155.8 (1P,s).

Example 31 Oxazaphospholidine monomer 5b

Compound 5b was obtained by using III-b instead of III-a in a similarmanner to compound 5a.

¹H NMR (300 MHz, CDCl₃) d 9.12 (1H, brs), 8.73 (1H, s), 8.24 (1H, s),8.07-8.01 (2H, m), 7.62-7.17 (22H, m), 6.83-6.77 (4H, m), 6.12 (1H, d,J=4.8 Hz), 4.84-4.73 (2H, m), 4.43 (1H, t, J=4.8 Hz), 4.25-4.19 (1H, m),3.77 (6H, s), 3.55-3.20 (4H, m), 3.28 (3H, s), 3.16-3.03 (1H, m),1.90-1.17 (6H, m), 0.65 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 155.0 (1P,s).

Example 32 Oxazaphospholidine monomer 6a

Compound 6a was obtained by using

“5′-O-(DMTr)-2′-O-methyl-4-N-(isobutyryl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.49 (1H, d, J=7.2 Hz), 7.58-7.20 (19H, m),6.96 (1H, d, J=7.2 Hz), 6.90-6.82 (4H, m), 5.98 (1H, s), 4.84 (1H, dd,J=13.1, 7.5 Hz), 4.59 (1H, dt, J=8.3, 4.5 Hz), 4.19-4.13 (1H, m), 3.79(6H, s), 3.78-3.72 (1H, m), 3.63-3.40 (3H, m), 3.55 (3H, s), 3.36-3.24(1H, m), 3.09-2.95 (1H, m), 2.59 (1H, septet, J=6.9 Hz), 1.85-1.53 (5H,m), 1.48-1.37 (1H, m), 1.24-1.17 (6H, m), 0.59 (3H, s); ³¹P NMR (121.5MHz, CDCl₃) d 155.2 (1P, s).

Example 33 Oxazaphospholidine monomer 6b

Compound 6b was obtained by using III-b instead of III-a in a similarmanner to compound 6a.

¹H NMR (300 MHz, CDCl₃) d 8.62 (1H, d, J=7.5 Hz), 7.57-7.23 (19H, m),7.02 (1H, d, J=7.5 Hz), 6.89-6.81 (4H, m), 5.92 (1H, s), 4.90 (1H, dt,J=9.0, 5.7 Hz), 4.61 (1H, dt, J=8.7, 4.8 Hz), 4.25-4.17 (1H, m), 3.81(6H, s), 3.67 (1H, d, J=4.5 Hz), 3.62-3.25 (4H, m), 3.38 (3H, s),3.16-3.02 (1H, m), 2.58 (1H, septet, J=6.9 Hz), 1.87-1.40 (6H, m),1.26-1.14 (6H, m), 0.64 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 158.2 (1P,s).

Example 34 Oxazaphospholidine monomer 7a

Compound 7a was obtained by using

“5′-O-(DMTr)-T-O-methyl-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.67 (1H, brs), 8.01 (1H, s), 7.56-7.16 (24H,m), 6.83-6.74 (4H, m), 6.08 (1H, d, J=6.9 Hz), 4.85-4.76 (1H, m), 4.84(2H, t, J=6.6 Hz), 4.65-4.56 (1H, m), 4.59 (2H, brs), 4.48 (1H, dd,J=6.6, 5.1 Hz), 4.09-4.05 (1H, m), 3.75 (6H, s), 3.60-3.42 (2H, m),3.40-3.26 (2H, m), 3.35 (3H, s), 3.18-3.05 (1H, m), 3.08 (2H, t, J=6.6Hz), 1.89-1.49 (3H, m), 1.48-1.16 (3H, m), 0.59 (3H, s); ³¹P NMR (121.5MHz, CDCl₃) d 156.9 (1P, s).

Example 35 Oxazaphospholidine monomer 7b

Compound 7b was obtained by using III-b instead of III-a in a similarmanner to compound 7a.

¹H NMR (300 MHz, CDCl₃) d 8.74 (1H, brs), 8.09 (1H, s), 7.56-6.94 (24H,m), 6.84-6.71 (4H, m), 6.09 (1H, d, J=4.8 Hz), 4.83-4.70 (2H, m), 4.83(2H, t, J=6.6 Hz), 4.63 (2H, brs), 4.35 (1H, t, J=5.0 Hz), 4.23-4.16(1H, m), 3.75 (6H, s), 3.58-3.19 (4H, m), 3.32 (3H, s), 3.16-3.04 (1H,m), 3.07 (2H, t, J=6.6 Hz), 1.90-1.55 (3H, m), 1.48-1.15 (3H, m), 0.64(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 154.6 (1P, s).

Example 36 Oxazaphospholidine monomer 8a

Compound 8a was obtained by using “5′-O-(DMTr)-2′-O-(methyl)uridine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.91 (1H, d, J=7.8 Hz), 7.58-7.20 (19H, m),6.88-6.80 (4H, m), 5.96 (1H, d, J=3.3 Hz), 5.19 (1H, d, J=7.8 Hz),4.88-4.78 (1H, m), 4.66-4.57 (1H, m), 4.03-3.95 (1H, m), 3.90-3.74 (1H,m), 3.78 (6H, s), 3.77-3.71 (1H, m), 3.58-3.29 (2H, m), 3.45 (3H, s),3.13-2.82 (2H, m), 1.88-1.53 (3H, m), 1.49-1.16 (3H, m), 0.60 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) d 155.3 (1P, s).

Example 37 Oxazaphospholidine monomer 8b

Compound 8b was obtained by using III-b instead of III-a in a similarmanner to compound 8a.

¹H NMR (300 MHz, CDCl₃) d 8.10 (1H, d, J=8.4 Hz), 7.58-7.20 (19H, m),6.87-6.79 (4H, m), 5.89 (1H, d, J=1.5 Hz), 5.21 (1H, d, J=8.4 Hz),4.92-4.82 (1H, m), 4.73-4.63 (1H, m), 4.15-4.08 (1H, m), 3.89-3.73 (1H,m), 3.78 (6H, s), 3.66-3.62 (1H, m), 3.57-3.27 (2H, m), 3.30 (3H, s),3.17-2.82 (2H, m), 1.89-1.55 (3H, m), 1.55-1.40 (1H, m), 1.35-1.15 (2H,m), 0.66 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.5 (1P, s).

Example 38 Oxazaphospholidine monomer 9a

Compound 9a was obtained by using

“5′-O-(DMTr)-2′-deoxy-2′-fluoro-6-N-(benzoyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.64 (1H, s), 8.14 (1H, s), 8.06-8.01 (2H, m),7.63-7.07 (23H, m), 6.78-6.70 (4H, m), 6.12 (1H, dd, J=18.0, 2.4 Hz),5.24-5.01 (2H, m), 4.94-4.84 (1H, m), 4.17-4.06 (1H, m), 3.73 (6H, s),3.55-3.40 (3H, m), 3.30-3.22 (1H, m), 3.03-2.88 (1H, m), 1.92-1.19 (6H,m), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 150.5 (1P, d, J=7.7 Hz).

Example 39 Oxazaphospholidine monomer 9b

Compound 9b was obtained by using III-b instead of III-a in a similarmanner to compound 9a.

¹H NMR (300 MHz, CDCl₃) d 9.07 (1H, brs), 8.80 (1H, s), 8.24 (1H, s),8.08-8.01 (2H, m), 7.66-7.15 (22H, m), 6.81-6.75 (4H, m), 6.14 (1H, dd,J=18.0, 1.8 Hz), 5.16-4.91 (3H, m), 4.28-4.21 (1H, m), 3.76 (6H, s),3.57-3.11 (5H, m), 1.82-1.16 (6H, m), 0.65 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) d 157.8 (1P, d, J=5.6 Hz).

Example 40 Oxazaphospholidine monomer 10a

Compound 10a was obtained by using

“5′-O-(DMTr)-2′-deoxy-2′-fluoro-4-N-(isobutyryl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.66 (1H, brs), 8.41 (1H, d, J=7.5 Hz),7.55-7.20 (19H, m), 7.01 (1H, d, J=7.5 Hz), 6.89-6.81 (4H, m), 6.06 (1H,d, J=15.9 Hz), 4.85 (1H, dd, J=51.4, 3.9 Hz), 4.84 (1H, dd, J=12.9, 7.5Hz), 4.77-4.59 (1H, m), 4.15-4.08 (1H, m), 3.79 (6H, s), 3.63-3.29 (4H,m), 3.10-2.96 (1H, m), 2.65 (1H, septet, J=6.9 Hz), 1.85-1.53 (3H, m),1.48-1.17 (3H, m), 1.21 (3H, d, J=4.8 Hz), 1.19 (3H, d, J=4.8 Hz), 0.59(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 155.5 (1P, d, J=6.6 Hz).

Example 41 Oxazaphospholidine monomer 10b

Compound 10b was obtained by using III-b instead of III-a in a similarmanner to compound 10a.

¹H NMR (300 MHz, CDCl₃) d 8.53 (1H, d, J=7.5 Hz), 7.57-7.23 (20H, m),7.10 (1H, d, J=7.5 Hz), 6.89-6.81 (4H, m), 6.10 (1H, d, J=15.9 Hz),5.00-4.92 (1H, m), 4.84 (1H, dd, J=51.5, 3.3 Hz), 4.75-4.58 (1H, m),4.24 (1H, d, J=9.3 Hz), 3.81 (6H, s), 3.65-3.39 (3H, m), 3.32-3.06 (2H,m), 2.59 (1H, septet, J=6.9 Hz), 1.88-1.53 (4H, m), 1.49-1.34 (2H, m),1.27-1.18 (6H, m), 0.65 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 159.0 (1P,d, J=4.4).

Example 42 Oxazaphospholidine monomer 11a

Compound 11a was obtained by using“5′-O-(DMTr)-T-deoxy-T-fluoro-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”instead of “5′-O— (DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.74 (1H, brs), 8.03 (1H, s), 7.55-6.94 (24H,m), 6.80-6.69 (4H, m), 6.21 (1H, dd, J=14.9, 3.6 Hz), 5.34 (1H, dt,J=52.3, 3.6 Hz), 5.01-4.75 (2H, m), 4.84 (1H, t, J=6.6 Hz), 4.62 (2H,brs), 4.15-4.07 (1H, m), 3.73 (6H, s), 3.59-3.29 (4H, m), 3.15-3.00 (1H,m), 3.07 (2H, t, J=6.6 Hz), 1.90-1.49 (3H, m), 1.47-1.12 (3H, m), 0.58(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 155.6 (1P, d, J=10.9 Hz).

Example 43 Oxazaphospholidine monomer 11b

Compound 11b was obtained by using III-b instead of III-a in a similarmanner to compound 11a.

¹H NMR (300 MHz, CDCl₃) d 8.81 (1H, brs), 8.06 (1H, s), 7.55-6.95 (24H,m), 6.77-6.69 (4H, m), 6.06 (1H, d, J=17.1 Hz), 5.24-5.08 (1H, m),5.04-4.80 (2H, m), 4.87 (1H, t, J=6.6 Hz), 4.62 (2H, brs), 4.25-4.19(1H, m), 3.73 (6H, s), 3.58-3.02 (5H, m), 3.10 (2H, t, J=6.6 Hz),1.90-1.56 (3H, m), 1.50-1.15 (3H, m), 0.63 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) d 158.0 (1P, d, J=4.4 Hz).

Example 44 Oxazaphospholidine monomer 12a

Compound 12a was obtained by using“5′-O-(DMTr)-2′-deoxy-2′-fluorouridine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.85 (1H, d, J=8.1 Hz), 7.58-7.20 (19H, m),6.87-6.79 (4H, m), 5.98 (1H, d, J=16.5 Hz), 5.23 (1H, d, J=8.1 Hz),4.86-4.61 (3H, m), 3.99 (1H, d, J=6.9 Hz), 3.76 (6H, d, J=3.0 Hz),3.56-3.34 (4H, m), 3.10-2.96 (1H, m), 1.88-1.74 (1H, m), 1.72-1.52 (2H,m), 1.48-1.16 (3H, m), 0.61 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 154.3(1P, d, J=8.9 Hz).

Example 45 Oxazaphospholidine monomer 12b

Compound 12b was obtained by using III-b instead of III-a in a similarmanner to compound 12a.

¹H NMR (300 MHz, CDCl₃) d 8.01 (1H, d, J=8.4 Hz), 7.58-7.20 (19H, m),6.87-6.79 (4H, m), 6.03 (1H, d, J=16.2 Hz), 5.29 (1H, d, J=8.4 Hz), 4.96(1H, dd, J=13.1, 7.5 Hz), 4.80-4.54 (2H, m), 4.15 (1H, d, J=9.0 Hz),3.78 (6H, s), 3.61-3.39 (3H, m), 3.37-3.25 (1H, m), 3.23-3.09 (1H, m),1.91-1.56 (3H, m), 1.51-1.13 (3H, m), 0.66 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) d 158.9 (1P, d, J=4.4 Hz).

Example 46 Oxazaphospholidine monomer 13a

Compound 13a was obtained by using

“5′-O-(DMTr)-2′-O-TOM-6-N-(acetyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.82 (1H, brs), 8.49 (1H, s), 8.10 (1H, s),7.58-7.17 (19H, m), 6.83-6.73 (4H, m), 6.11 (1H, d, J=6.6 Hz), 5.15 (1H,dd, J=6.6, 5.4 Hz), 4.98-4.77 (4H, m), 4.18-4.11 (1H, m), 3.76 (6H, s),3.59-3.25 (4H, m), 3.16-3.02 (1H, m), 2.62 (3H, s), 1.91-1.53 (3H, m),1.49-1.18 (3H, m), 0.96-0.80 (3H, m), 0.90 (18H, s), 0.62 (3H, s); ³¹PNMR (121.5 MHz, CDCl₃) d 156.7 (1P, s).

Example 47 Oxazaphospholidine monomer 13b

Compound 13b was obtained by using III-b instead of III-a in a similarmanner to compound 13a.

¹H NMR (300 MHz, CDCl₃) d 8.56 (1H, brs), 8.55 (1H, s), 8.13 (1H, s),7.57-7.17 (19H, m), 6.82-6.73 (4H, m), 6.16 (1H, d, J=5.7 Hz), 5.06 (1H,t, J=5.6 Hz), 4.93 (1H, d, J=5.1 Hz), 4.83 (1H, d, J=5.1 Hz), 4.81-4.69(2H, m), 4.27-4.19 (1H, m), 3.76 (6H, s), 3.55-3.40 (2H, m), 3.33-3.16(2H, m), 3.12-2.97 (1H, m), 2.63 (3H, s), 1.88-1.52 (3H, m), 1.45-1.16(3H, m), 0.91-0.79 (3H, m), 0.86 (18H, s), 0.64 (3H, s); ³¹P NMR (121.5MHz, CDCl₃) d 154.8 (1P, s).

Example 48 Oxazaphospholidine monomer 14a

Compound 14a was obtained by using“5′-O-(DMTr)-2′-O-TOM-4-N-(acetyl)cytidine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 10.04 (1H, brs), 8.30 (1H, d, J=7.5 Hz),7.51-7.21 (19H, m), 6.99 (1H, d, J=7.5 Hz), 6.89-6.81 (4H, m), 6.12 (1H,d, J=3.3 Hz), 5.07 (1H, d, J=4.8 Hz), 5.05 (1H, d, J=4.8 Hz), 4.84-4.75(1H, m), 4.62-4.52 (1H, m), 4.31-4.25 (1H, m), 4.08-4.01 (1H, m), 3.78(6H, d, J=3.0 Hz), 3.55-3.23 (4H, m), 3.10-2.96 (1H, m), 2.24 (3H, s),1.84-1.49 (3H, m), 1.46-0.96 (24H, m), 0.58 (3H, s); ³¹P NMR (121.5 MHz,CDCl₃) d 156.5 (1P, s).

Example 49 Oxazaphospholidine monomer 14b

Compound 14b was obtained by using III-b instead of III-a in a similarmanner to compound 14a.

¹H NMR (300 MHz, CDCl₃) d 10.19 (1H, brs), 8.46 (1H, d, J=7.5 Hz),7.54-7.23 (19H, m), 7.01 (1H, d, J=7.5 Hz), 6.88-6.79 (4H, m), 6.19 (1H,d, J=1.8 Hz), 5.11 (1H, d, J=4.8 Hz), 5.07 (1H, d, J=4.8 Hz), 4.81-4.71(1H, m), 4.60-4.51 (1H, m), 4.26-4.18 (2H, m), 3.79 (6H, s), 3.63-3.55(1H, m), 3.48-3.28 (2H, m), 3.21-2.94 (2H, m), 2.26 (3H, s), 1.81-1.49(3H, m), 1.43-0.96 (24H, m), 0.62 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d156.4 (1P, s).

Example 50 Oxazaphospholidine monomer 15a

Compound 15a was obtained by using

“5′-O-(DMTr)-2′-O-TOM-2-N-(acetyl)guanosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.70 (1H, s), 7.63-7.13 (21H, m), 6.84-6.76(4H, m), 5.77 (1H, d, J=8.4 Hz), 5.41-5.33 (1H, m), 4.90 (2H, s),4.78-4.68 (2H, m), 3.86 (1H, brs), 3.75 (3H, s), 3.74 (3H, s), 3.56-3.41(2H, m), 3.32-2.90 (3H, m), 1.92-1.10 (9H, m), 0.97-0.87 (21H, m), 0.52(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 158.1 (1P, s).

Example 51 Oxazaphospholidine monomer 15b

Compound 15b was obtained by using III-b instead of III-a in a similarmanner to compound 15a.

¹H NMR (300 MHz, CDCl₃) d 7.77 (1H, s), 7.56-7.15 (21H, m), 6.82-6.75(4H, m), 5.86 (1H, d, J=7.5 Hz), 5.26-5.17 (1H, m), 4.95 (1H, d, J=5.4Hz), 4.85 (1H, d, J=5.4 Hz), 4.78-4.71 (1H, m), 4.59-4.49 (1H, m),4.10-4.05 (1H, m), 3.74 (6H, s), 3.52-3.37 (2H, m), 3.30-3.18 (1H, m),3.11-2.85 (2H, m), 1.85-1.15 (9H, m), 0.93-0.84 (21H, m), 0.62 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) d 152.3 (1P, s).

Example 52 Oxazaphospholidine monomer 16a

Compound 16a was obtained by using “5′-O-(DMTr)-2′-O-TOM-uridine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.76 (1H, d, J=8.1 Hz), 7.55-7.18 (20H, m),6.88-6.80 (4H, m), 6.11 (1H, d, J=6.0 Hz), 5.32 (1H, d, J=8.1 Hz), 4.99(1H, d, J=5.1 Hz), 4.93 (1H, d, J=5.1 Hz), 4.84-4.75 (1H, m), 4.54-4.46(1H, m), 4.38 (1H, t, J=5.7 Hz), 3.87-3.83 (1H, m), 3.78 (3H, s), 3.77(3H, s), 3.56-3.42 (1H, m), 3.39-3.28 (1H, m), 3.36 (1H, dd, J=11.0, 2.7Hz), 3.25 (1H, dd, J=11.0, 2.7 Hz), 3.16-3.03 (1H, m), 1.88-1.12 (6H,m), 1.08-0.97 (21H, m), 0.59 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 156.6(1P, s).

Example 53 Oxazaphospholidine monomer 16b

Compound 16b was obtained by using III-b instead of III-a in a similarmanner to compound 16a.

¹H NMR (600 MHz, CDCl₃) d 7.87 (1H, d, J=7.8 Hz), 7.52-7.48 (4H, m),7.38-7.21 (16H, m), 6.83-6.79 (4H, m), 6.14 (1H, d, J=4.8 Hz), 5.33 (1H,d, J=7.8 Hz), 4.99 (1H, d, J=5.4 Hz), 4.89 (1H, d, J=5.4 Hz), 4.67 (1H,dd, J=13.8, 7.2 Hz), 4.52 (1H, dt, J=10.4, 4.8 Hz), 4.31 (1H, t, J=4.8Hz), 4.06-4.03 (1H, m), 3.78 (3H, s), 3.77 (3H, s), 3.47 (1H, dd,J=10.4, 2.4 Hz), 3.47-3.39 (1H, m), 3.22-3.17 (2H, m), 3.00 (1H, ddd,J=19.5, 10.4, 4.8 Hz), 1.82-1.74 (1H, m), 1.68-1.58 (1H, m), 1.56 (1H,dd, J=14.4, 8.4 Hz), 1.38 (1H, dd, J=14.4, 7.2 Hz), 1.31-1.25 (1H, m),1.26-1.17 (1H, m), 1.08-0.98 (21H, m), 0.63 (3H, s); ³¹P NMR (243.0 MHz,CDCl₃) d 154.3 (1P, s).

Example 54 Oxazaphospholidine monomer 17a

Compound 17a was obtained by using

“5′-O-(DMTr)-2′-0,4′-C-methylene-6-N-(benzoyl)adenosine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 9.10 (1H, brs), 8.76 (1H, s), 8.32 (1H, s),8.04 (2H, d, J=7.2 Hz), 7.64-7.18 (22H, m), 6.84 (4H, d, J=8.7 Hz), 6.10(1H, s), 4.76 (1H, d J=6.9 Hz), 4.58 (1H, s), 4.61-4.51 (1H, m), 3.91(1H, d, J=7.8 Hz), 3.77 (1H, d, J=7.8 Hz), 3.75 (6H, s), 3.50 (1H, s),3.47-3.33 (1H, m), 3.31-3.19 (1H, m), 3.03-2.88 (1H, m), 1.84-1.09 (6H,m), 0.51 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 152.9 (1P, s).

Example 55 Oxazaphospholidine monomer 17b

Compound 17b was obtained by using III-b instead of III-a in a similarmanner to compound 17a.

¹H NMR (300 MHz, CDCl₃) d 8.81 (1H, s), 8.30 (1H, s), 8.07-8.00 (2H, m),7.64-7.17 (22H, m), 6.86-6.79 (4H, m), 6.12 (1H, s), 4.81-4.72 (1H, m),4.62 (1H, d J=7.2 Hz), 4.57 (1H, s), 3.94 (1H, d, J=7.8 Hz), 3.89 (1H,d, J=7.8 Hz), 3.77 (6H, s), 3.48 (2H, s), 3.46-3.32 (1H, m), 3.24-3.13(1H, m), 3.10-2.97 (1H, m), 1.84-1.49 (3H, m), 1.42-1.09 (3H, m), 0.58(3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.3 (1P, s).

Example 56 Oxazaphospholidine monomer 18a

Compound 18a was obtained by using

“5′-O-(DMTr)-T-0,4′-C-methylene-4-N-(isobutyryl)-5-methylcytidine”instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.88 (1H, brs), 7.58-7.18 (20H, m), 6.88-6.80(4H, m), 5.65 (1H, s), 4.69-4.60 (1H, m), 4.52 (1H, d, J=6.6 Hz), 4.49(1H, s), 3.81-3.74 (1H, m), 3.75 (3H, s), 3.73 (3H, s), 3.64 (1H, d,J=8.1 Hz), 3.56 (1H, d, J=11.1 Hz), 3.53 (1H, d, J=8.1 Hz), 3.46 (1H, d,J=11.1 Hz), 3.56-3.40 (1H, m), 3.32-3.20 (1H, m), 3.14-3.00 (1H, m),1.85-1.12 (6H, m), 1.60 (3H, s), 1.19 (6H, d, J=6.9 Hz), 0.55 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) d 155.9 (1P, s).

Example 57 Oxazaphospholidine monomer 18b

Compound 18b was obtained by using III-b instead of III-a in a similarmanner to compound 18a.

¹H NMR (300 MHz, CDCl₃) d 7.86 (1H, brs), 7.56-7.19 (20H, m), 6.88-6.79(4H, m), 5.69 (1H, s), 4.86-4.76 (1H, m), 4.46 (1H, s), 4.45 (1H, d,J=7.5 Hz), 3.80-3.75 (1H, m), 3.79 (6H, s), 3.74 (1H, d, J=8.1 Hz), 3.69(1H, d, J=8.1 Hz), 3.51 (1H, d, J=11.1 Hz), 3.44-3.30 (1H, m), 3.39 (1H,d, J=11.1 Hz), 3.29-3.17 (1H, m), 3.11-2.97 (1H, m), 1.86-1.52 (3H, m),1.64 (3H, s), 1.45-1.10 (3H, m), 1.21 (6H, d, J=6.6 Hz), 0.62 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) d 158.2 (1P, s).

Example 58 Oxazaphospholidine monomer 19a

Compound 19a was obtained by using

“5′-O-(DMTr)-T-0,4′-C-methylene-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”instead of “5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine”in a similar manner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 8.71 (1H, brs), 8.16 (1H, s), 7.50-7.17 (21H,m), 7.09-7.01 (3H, m), 6.86-6.79 (4H, m), 6.03 (1H, s), 4.84 (2H, t,J=6.6 Hz), 4.72 (2H, s), 4.68 (1H, d, J=7.2 Hz), 4.55-4.46 (1H, m), 4.50(1H, s), 3.90 (1H, d, J=7.8 Hz), 3.77 (1H, d, J=7.8 Hz), 3.75 (6H, s),3.51 (1H, d, J=10.8 Hz), 3.47 (1H, d, J=10.8 Hz), 3.45-3.21 (2H, m),3.08 (2H, t, J=6.6 Hz), 3.03-2.89 (1H, m), 1.80-1.08 (6H, m), 0.47 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) d 153.2 (1P, s).

Example 59 Oxazaphospholidine monomer 19b

Compound 19b was obtained by using III-b instead of III-a in a similarmanner to compound 19a.

¹H NMR (300 MHz, CDCl₃) d 8.86 (1H, brs), 8.13 (1H, s), 7.55-7.17 (21H,m), 7.08-6.98 (3H, m), 6.95-6.78 (4H, m), 6.01 (1H, s), 4.86 (2H, t,J=6.6 Hz), 4.82-4.73 (1H, m), 4.70 (2H, s), 4.64 (1H, d, J=7.5 Hz), 4.49(1H, s), 3.94 (1H, d, J=7.8 Hz), 3.89 (1H, d, J=7.8 Hz), 3.77 (6H, s),3.46 (2H, s), 3.45-3.30 (1H, m), 3.24-3.12 (1H, m), 3.09 (2H, t, J=6.6Hz), 3.09-2.96 (1H, m), 1.81-1.50 (3H, m), 1.41-1.06 (3H, m), 0.58 (3H,s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.4 (1P, s).

Example 60 Oxazaphospholidine monomer 20a

Compound 20a was obtained by using

“5′-O-(DMTr)-2′-0,4′-C-methylene-5-methyluridine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.71 (1H, d, J=0.9 Hz), 7.50-7.17 (20H, m),6.87-6.80 (4H, m), 5.61 (1H, s), 4.69-4.60 (1H, m), 4.55 (1H, d, J=6.9Hz), 4.41 (1H, s), 3.74 (3H, s), 3.73 (3H, s), 3.64 (1H, d, J=7.8 Hz),3.55 (1H, d, J=7.8 Hz), 3.53 (1H, d, J=10.8 Hz), 3.46 (1H, d, J=10.8Hz), 3.56-3.42 (1H, m), 3.35-3.24 (1H, m), 3.13-3.00 (1H, m), 1.85-1.45(3H, m), 1.55 (3H, d, J=0.9 Hz), 1.41-1.12 (3H, m), 0.56 (3H, s); ³¹PNMR (121.5 MHz, CDCl₃) d 155.1 (1P, s).

Example 61 Oxazaphospholidine monomer 20b

Compound 20b was obtained by using III-b instead of III-a in a similarmanner to compound 20a.

¹H NMR (300 MHz, CDCl₃) d 7.69 (1H, s), 7.56-7.19 (20H, m), 6.88-6.79(4H, m), 5.66 (1H, s), 4.87-4.77 (1H, m), 4.47 (1H, d, J=7.8 Hz), 4.40(1H, s), 3.78 (6H, s), 3.74 (1H, d, J=7.8 Hz), 3.68 (1H, d, J=7.8 Hz),3.50 (1H, d, J=10.8 Hz), 3.46-3.32 (1H, m), 3.39 (1H, d, J=10.8 Hz),3.30-3.19 (1H, m), 3.12-2.98 (1H, m), 1.85-1.56 (3H, m), 1.59 (3H, s),1.46-1.12 (3H, m), 0.63 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 158.1 (1P,s).

Example 62 Oxazaphospholidine monomer 21n

Compound 21a was obtained by using

“5′-O-(DMTr)-2′-O-methoxyethyl-5-methyluridine” instead of“5′-O-(DMTr)-2-N-(phenoxyacetyl)-6-O-(cyanoethyl)guanosine” in a similarmanner to compound 3a.

¹H NMR (300 MHz, CDCl₃) d 7.62-7.18 (21H, m), 6.84 (4H, d, J=8.7 Hz),6.07 (1H, d, J=5.7 Hz), 4.86-4.76 (1H, m), 4.63-4.54 (1H, m), 4.20 (1H,t, J=5.4 Hz), 3.95-3.89 (1H, m), 3.78 (6H, s), 3.78-3.71 (2H, m),3.60-3.48 (2H, m), 3.44-3.02 (5H, m), 3.31 (3H, s), 1.88-1.15 (6H, m),1.35 (3H, s), 0.58 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 156.3 (1P, s).

Example 63 Oxazaphospholidine monomer 21b

Compound 21b was obtained by using III-b instead of III-a in a similarmanner to compound 21a.

¹H NMR (300 MHz, CDCl₃) d 7.71 (1H, d, J=1.2 Hz), 7.55-7.22 (20H, m),6.86-6.78 (4H, m), 5.99 (1H, d, J=3.9 Hz), 4.78-4.62 (2H, m), 4.13-4.08(1H, m), 4.07-4.02 (1H, m), 3.77 (6H, s), 3.77-3.70 (1H, m), 3.65-3.56(1H, m), 3.52-3.36 (4H, m), 3.33-3.14 (2H, m), 3.29 (3H, s), 3.08-2.94(1H, m), 1.86-1.72 (1H, m), 1.71-1.55 (2H, m), 1.30 (3H, d, J=1.2 Hz),1.47-1.16 (3H, m) 0.64 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 155.6 (1P,s).

Example 64 Oxazaphospholidine monomer 22a

Compound 22a was obtained by using VII-a instead of III-a in a similarmanner to compound 4a.

¹H NMR (300 MHz, CDCl₃) d 7.57 (1H, d, J=0.9 Hz), 7.37-6.94 (20H, m),6.87-6.78 (4H, m), 6.48 (1H, dd, J=8.6, 5.7 Hz), 5.42 (1H, dd, J=11.0,5.1 Hz), 4.81-4.71 (1H, m), 4.02 (1H, d, J=11.0 Hz), 3.83 (1H, d, J=2.1Hz), 3.79 (6H, s), 3.61-3.41 (2H, m), 3.24-3.09 (1H, m), 3.16 (1H, dd,J=10.8, 2.4 Hz), 3.02 (1H, dd, J=10.8, 2.4 Hz), 2.54-2.44 (1H, m),2.34-2.22 (1H, m), 1.94-1.79 (1H, m), 1.74-1.56 (1H, m), 1.38 (3H, s),1.38-1.28 (2H, m); ³¹P NMR (121.5 MHz, CDCl₃) d 160.9 (1P, s).

Example 65 Oxazaphospholidine monomer 22b

Compound 22b was obtained by using VII-b instead of VII-a in a similarmanner to compound 22a.

¹H NMR (300 MHz, CDCl₃) d 7.57 (1H, d, J=1.5 Hz), 7.43-7.11 (20H, m),6.85-6.78 (4H, m), 6.48 (1H, dd, J=7.5, 5.7 Hz), 5.58 (1H, dd, J=11.4,5.1 Hz), 4.82-4.73 (1H, m), 4.17-4.02 (2H, m), 3.78 (6H, s), 3.56-3.40(3H, m), 3.32 (1H, dd, J=10.7, 2.4 Hz), 3.22-3.07 (1H, m), 2.26-2.04(2H, m), 1.95-1.81 (1H, m), 1.74-1.56 (1H, m), 1.40 (3H, d, J=1.5 Hz),1.44-1.34 (2H, m); ³¹P NMR (121.5 MHz, CDCl₃) d 162.2 (1P, s).

Example 66 Oxazaphospholidine monomer 23a

Compound 23a was obtained by using IX-a instead of III-a in a similarmanner to compound 4a.

¹H NMR (300 MHz, CDCl₃) d 9.22 (1H, brs), 8.05-7.99 (2H, m), 7.52 (1H,d, J=1.2 Hz), 7.41-7.19 (11H, m), 6.87-6.79 (4H, m), 6.37 (1H, dd,J=8.4, 5.7 Hz), 4.88-4.75 (2H, m), 3.86-3.80 (1H, m), 3.79 (6H, s),3.64-3.49 (2H, m), 3.27-3.12 (3H, m), 2.97 (2H, d, J=6.6 Hz), 2.51-2.41(1H, m), 2.33-2.20 (1H, m), 2.03-1.75 (2H, m), 1.72-1.59 (1H, m),1.46-1.36 (1H, m), 1.40 (3H, s); ³¹P NMR (121.5 MHz, CDCl₃) d 157.5 (1P,s).

Example 67 Oxazaphospholidine monomer 23b

Compound 23b was obtained by using IX-b instead of IX-a in a similarmanner to compound 23a.

¹H NMR (300 MHz, CDCl₃) d 8.67 (1H, brs), 8.18-8.11 (2H, m), 7.57 (1H,d, J=1.2 Hz), 7.47-7.22 (11H, m), 6.86-6.79 (4H, m), 6.29 (1H, t, J=6.6Hz), 4.87 (1H, dt, J=7.5, 5.7 Hz), 4.80-4.72 (1H, m), 4.11-4.05 (1H, m),3.79 (6H, s), 3.67-3.47 (2H, m), 3.43 (1H, dd, J=10.8, 2.7 Hz), 3.27(1H, dd, J=10.8, 2.4 Hz), 3.25-3.13 (1H, m), 3.07-2.99 (2H, m),2.19-2.12 (2H, m), 2.03-1.62 (3H, m), 1.46-1.30 (1H, m), 1.41 (3H, s);³¹P NMR (121.5 MHz, CDCl₃) d 158.1 (1P, s).

Example 68 Oxazaphospholidine monomer 24a

Compound 24a was obtained by using XIII-a instead of III-a in a similarmanner to compound 4a.

¹H NMR (600 MHz, CDCl₃) d 7.76 (2H, d, J=9.0 Hz), 7.62 (1H, d, J=1.2Hz), 7.40 (2H, d, J=7.2 Hz), 7.32-7.23 (10H, m), 6.85 (4H, d, J=8.4 Hz),6.41 (1H, dd, J=8.4, 5.4 Hz), 4.94 (1H, dd, J=12.3, 5.4 Hz), 4.84-4.79(1H, m), 4.03-4.01 (1H, m), 3.79 (6H, s), 3.59-3.53 (1H, m), 3.52-3.44(2H, m), 3.41 (1H, dd, J=14.7, 7.2 Hz), 3.37-3.30 (2H, m), 3.13 (1H,ddd, J=19.3, 10.3, 4.1 Hz), 2.50-2.44 (1H, m), 2.39 (3H, s), 2.35-2.29(1H, m), 1.91-1.72 (2H, m), 1.64-1.59 (1H, m), 1.40 (3H, s), 1.12-1.05(1H, m); ³¹P NMR (243.0 MHz, CDCl₃) d 154.2 (1P, s).

General Procedure for the Synthesis of Chrial-Oligos:

The automated solid-phase synthesis of chiral-oligos were performedaccording to the cycles shown in Table 1. After the synthesis, the resinwas treated with a 25% NH₃ aqueous solution (1 mL) for 12 h at 55degrees C. The mixture was cooled to room temperature and the resin wasremoved by membrane filtration. The filtrate was concentrated to drynessunder reduced pressure. The residue was dissolved in H₂O (3 mL) andanalyzed by RP-UPLC-MS with a linear gradient of acetonitrile (0-50%/30min) in 0.1 M triethylammonium acetate buffer (pH 7.0) at 50 degrees C.at a rate of 0.3 mL/min.

TABLE 1 step operation reagents and solvent volume waiting 1detritylation 3% DCA/DCM 1.6 mL 20 s 2 coupling 0.1M monomer/MeCN + 0.5mL 5 min 1M 3 capping Ac₂O/THF-pyridine + 0.5 mL 30 s 16%/THF 4oxidation/ 0.5M CSO/MeCN or 0.5 mL 90 s urization 0.1M MeCN

Comparison Example 1

The above Compound 25, which represents a conventional monomer, was usedto produce oligos. FIG. 2 shows a chart of products obtained throughComparison Example 1.

Analysis

The monomers of the working examples were chemically stable. The isolateyield of the monomers were more than 80%, which was higher that ofconventional method.

We synthesized oligonucleotide derivatives using the chiral reagents ofthe above working examples based on the second general procedure andmonomers of the above working examples based on the first generalprocedure. As shown in FIG. 2, the conventional monomer causesincomplete de-protection products, side products and failure sequences.On the other hand, the method of the invention causes little incompletede-protection products and little side products even though it causesfailure sequences as shown in FIG. 1. It is obvious that the method ofthe invention can lessen the incomplete de-protection products and sideproducts. It was easy to isolate the targeted oligonucleotidederivatives because the present invention can lessen undesirableproducts.

1. A chiral reagent or a salt thereof, the chiral reagent havingfollowing chemical formula (I),

wherein G¹ and G² are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group, a group of formula (II), (III) or (V), orboth G¹ and G² taken together to form a group of formula (IV),

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G³¹ to G³³ are independently C₁₋₄ alkyl group, C₁₋₄ alkoxygroup, C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ arylgroup, C 1-4 alkoxy C₆₋₁₄ alkyl group, aryl group, or C₆₋₁₄ aryl C₁₋₄

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group, G³ and G⁴ areindependently a hydrogen atom, C₁₋₃ alkyl group, C₆₋₁₄ aryl group, orboth G³ and G⁴ taken together to form a heteroatom-containing ring thathas 3 to 16 carbon atoms,

wherein G⁵¹ to G⁵³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group, C₁₋₃ alkyl group or C₁₋₃ alkyloxy group. 2.The chiral reagent or a salt thereof in accordance with claim 1, whereinthe chiral reagent has following chemical formula (I′).


3. The chiral reagent or a salt thereof in accordance with claim 2,wherein each of G¹ and G² is a group of formula (II), wherein G²¹ to G²³are independently a hydrogen atom, a nitro group, a halogen atom, acyano group or C₁₋₃ alkyl group.
 4. The chiral reagent or a salt thereofin accordance with claim 2, wherein each of G¹ and G² is a group offormula (II), wherein each of G²¹ to G²³ is a hydrogen atom.
 5. Thechiral reagent or a salt thereof in accordance with claim 2, wherein G¹is a hydrogen atom, and G² is a group of formula (II), wherein G²¹ toG²³ are independently a hydrogen atom, a nitro group, a halogen atom, acyano group or C₁₋₃ alkyl group.
 6. The chiral reagent or a salt thereofin accordance with claim 2, wherein G¹ is a hydrogen atom and G² is agroup of formula (II), wherein each of G²¹ and G²² is a hydrogen atomand G²³ is a nitro group.
 7. The chiral reagent or a salt thereof inaccordance with claim 2, wherein G¹ is a hydrogen atom, and G² is agroup of formula (III), wherein G³¹ to G³³ are independently C₁₋₄ alkylgroup, C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ arylgroup, C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄ alkyl group. 8.The chiral reagent or a salt thereof in accordance with claim 2, whereinG¹ is a hydrogen atom, and G² is a group of formula (III), wherein G³¹to G³³ are independently C₁₋₄ alkyl group, C₆ aryl group, C₇₋₁₀ aralkylgroup, C₁₋₄ alkyl C₆ aryl group, C₁₋₄ alkoxy C₆ aryl group, or C₆ arylC₁₋₄ alkyl group.
 9. The chiral reagent or a salt thereof in accordancewith claim 2, wherein G¹ is a hydrogen atom and G² is a group of formula(III), wherein G³¹ to G³³ are independently C₁₋₄ alkyl group, or C₆ arylgroup.
 10. The chiral reagent or a salt thereof in accordance with claim2, wherein G¹ is a hydrogen atom and G² is a group of formula (III),wherein G³¹ to G³³ are independently C₁₋₄ alkyl group.
 11. The chiralreagent or a salt thereof in accordance with claim 2, wherein G¹ is ahydrogen atom and G² is a group of formula (III), wherein G³¹ and G³³are C₆ aryl group and G³² is C₁₋₄ alkyl group.
 12. The chiral reagent ora salt thereof in accordance with claim 2, wherein G¹ and G² takentogether to form a group of formula (IV), wherein G⁴¹ to G⁴⁶ areindependently a hydrogen atom, a nitro group, a halogen atom, a cyanogroup or C₁₋₃ alkyl group.
 13. The chiral reagent or a salt thereof inaccordance with claim 2, wherein G¹ and G² taken together to form agroup of formula (IV), wherein each of G⁴¹ to G⁴⁶ is a hydrogen atom.14. The chiral reagent or a salt thereof in accordance with claim 2,wherein G¹ is a hydrogen atom and G² is a group of formula (V), whereineach of G⁵¹ to G⁵³ are independently a hydrogen atom, a nitro group, amethyl group, or a methoxy group.
 15. The chiral reagent or a saltthereof in accordance with claim 2, wherein G¹ is a hydrogen atom and G²is a group of formula (V), wherein each of G⁵¹ and G⁵³ is a hydrogenatom and G⁵³ is a 4-methyl group.
 16. The chiral reagent or a saltthereof in accordance with claim 2, wherein the chiral reagent isrepresented by III-a, III-b, V-a, VII-a, VII-b, IX-a, IX-b, XI-a, XIII-aor XIII-b: (S)-2-(Methyldiphenylsilyl)-1-((S)-pyrrolidin-2-yl)ethanol(III-a) (R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol(III-b) (S)-2-(Trimethylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (V-a)(R)-2,2-Diphenyl-1-((S)-pyrrolidin-2-yl)ethanol (VII-a)(S)-2,2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (VII-b)(R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (IX-a)(S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (IX-b)(R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (XI-a)(S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (XIII-a)(R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (XIII-b)
 17. Anucleoside 3′-phosphoramidite derivative which is represented by formula(Va) or (Vb),

wherein G¹ and G² are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group, a group of formula (II), (III) or (V), bothG¹ and G² taken together to form a group of formula (IV),

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G³¹ to G³³ are independently C₁₋₄ alkyl group, C₁₋₄ alkoxygroup, C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄ alkyl C₆₋₁₄ arylgroup, C 1-4 alkoxy C₆₋₁₄ alkyl group, aryl group, or C₆₋₁₄ aryl C₁₋₄

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G⁵¹ to G⁵³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group, C₁₋₃ alkyl group or C₁₋₃ alkyloxy group, G³and G⁴ are independently a hydrogen atom, C₁₋₃ alkyl group, C₆₋₁₄ arylgroup, or both G³ and G⁴ taken together to form a heteroatom-containingring that has 3 to 16 carbon atoms. G⁵ is a protective group of ahydroxyl group, R² is hydrogen, —OH, —SH, —NR^(d)R^(d), —N₃, halogen,alkyl, alkenyl, alkynyl, alkyl-Y¹-, alkenyl-Y¹-, alkynyl-Y¹-, aryl-Y¹-,heteroaryl-Y¹-, —OR^(b), or —SR^(b), wherein R^(b) is a blocking moiety,Y¹ is O, NR^(d), S, or Se, R^(d) is independently hydrogen, alkyl,alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate,—P(O)(R^(e))₂, or —HP(O)(R^(e)), R^(e) is independently hydrogen, alkyl,aryl, alkenyl, alkynyl, alkyl-Y²—, alkenyl-Y²—, alkynyl-Y²—, aryl-Y²—,or heteroaryl-Y²—, or a cation which is Na⁺, Li⁺, or K⁺, Y² is O,NR^(d), or S, R³ is a group represented by —CH₂—, —(CH₂)₂—, —CH₂NH—, or—CH₂N(CH₃)—, and Bs is a group selected from the groups represented byfollowing formula (VI) to (XI) or derivatives thereof.


18. The nucleoside 3′-phosphoramidite derivative in accordance withclaim 17, wherein the nucleoside 3′-phosphoramidite derivative isrepresented by formula (Va′) or (Vb′).


19. The nucleoside 3′-phosphoramidite derivative in accordance withclaim 17, wherein the nucleoside 3′-phosphoramidite derivative isrepresented by formula 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b,7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, 11a, 11b, 12a, 12b, 13a, 13b, 14a,14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a,21b, 22a, 22b, 23a, 23b, or 24a.

wherein DMTr represents a 4,4′-dimethoxytrityl group and TOM representsa triisopropylsiloxymethyl group.
 20. A method for synthesis ofstereocontrolled phosphorus atom-modified oligonucleotide derivativescomprising steps of: reacting a molecule comprising an achiralH-phosphonate moiety, a chiral reagent or a salt thereof to form amonomer of a nucleoside 3′-phosphoramidite derivative, reacting themonomer and a nucleoside to form a condensed intermediate; andconverting the condensed intermediate to the nucleic acid comprising achiral X-phosphonate moiety, wherein the chiral reagent has followingchemical formula (I),

wherein G¹ and G² are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group, a group of formula (II), (III) or (V), orboth G¹ and G² taken together to form a group of formula (IV),

wherein G²¹ to G²³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G³¹ to G³³ are independently C₁₋₄ alkyl group or C₆₋₁₄ arylgroup, C₁₋₄ alkoxy group, C₆₋₁₄ aryl group, C₇₋₁₄ aralkyl group, C₁₋₄alkyl C₆₋₁₄ aryl group, C₁₋₄ alkoxy C₆₋₁₄ aryl group, or C₆₋₁₄ aryl C₁₋₄alkyl group,

wherein G⁴¹ to G⁴⁶ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group or C₁₋₃ alkyl group,

wherein G⁵¹ to G⁵³ are independently a hydrogen atom, a nitro group, ahalogen atom, a cyano group, C₁₋₃ alkyl group or C₁₋₃ alkyloxy group,and G³ and G⁴ are independently a hydrogen atom, C₁₋₃ alkyl group, C₆₋₁₄aryl group, or both G³ and G⁴ taken together to form aheteroatom-containing ring that has 3 to 16 carbon atoms.
 21. The methodfor synthesis of stereocontrolled phosphorus atom-modifiedoligonucleoside derivatives in accordance with claim 20, wherein thewherein the chiral reagent has following chemical formula (I′).


22. The method for synthesis of a stereocontrolled phosphorusatom-modified oligonucleotide derivative in accordance with claim 20,wherein the chiral reagent is represented by III-a, III-b, V-a, VII-a,VII-b, IX-a, IX-b, XI-a, XIII-a and XIII-b:(S)-2-(Methyldiphenylsilyl)-1-((S)-pyrrolidin-2-yl)ethanol (III-a)(R)-2-(Methyldiphenylsilyl)-1-((R)-1-pyrrolidin-2-yl)ethanol (III-b)(S)-2-(Trimethylsilyl)-1-((S)-1-pyrrolidin-2-yl)ethanol (V-a)(R)-2,2-Diphenyl-1-((S)-pyrrolidin-2-yl)ethanol (VII-a)(S)-2,2-Diphenyl-1-((R)-pyrrolidin-2-yl)ethanol (VII-b)(R)-2-(4-Nitrophenyl)-1-((S)-pyrrolidin-2-yl)ethanol (IX-a)(S)-2-(4-Nitrophenyl)-1-((R)-pyrrolidin-2-yl)ethanol (IX-b)(R)-(9H-Fluororen-9-yl)((S)-pyrrolidin-2-yl)methanol (XI-a)(S)-2-Tosyl-1-((S)-1-tritylpyrrolidin-2-yl)ethanol (XIII-a)(R)-2-Tosyl-1-((R)-1-tritylpyrrolidin-2-yl)ethanol (XIII-b)
 23. A methodfor synthesis of stereocontrolled phosphorus atom-modifiedoligonucleotide derivatives comprising steps of: reacting a nucleoside3′-phosphoramidite derivative which is represented by formula (Va) or(Vb), with an activating reagent and a nucleoside to form a condensedintermediate; and converting the condensed intermediate to the nucleicacid comprising a chiral X-phosphonate moiety.


24. The method for synthesis of stereocontrolled phosphorusatom-modified oligonucleotide derivatives in accordance with claim 23,wherein the nucleoside 3′-phosphoramidite derivative is represented byformula (Va′) or (Vb′).


25. The method for synthesis of stereocontrolled phosphorusatom-modified oligonucleotide derivatives in accordance with claim 23,wherein the nucleoside 3′-phosphoramidite derivative is represented byformula 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b,9a, 9b, 10a, 10b, 11a, 11b, 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a,16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a,23b, or 24a,

wherein DMTr represents a 4,4′-dimethoxytrityl group and TOM representsa triisopropylsiloxymethyl group.